Comparative cytogenetics and heterochromatic patterns in two species of the genus Acanthostracion (Ostraciidae: Tetraodontiformes)

Marine Genomics 4 (2011) 215–220

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Comparative cytogenetics and heterochromatic patterns in two species of the genus Acanthostracion (Ostraciidae: Tetraodontiformes) Pablo Ariel Martinez ⁎, Uedson Pereira Jacobina, Wagner Franco Molina Universidade Federal do Rio Grande do Norte (UFRN), Departamento de Biologia Celular e Genética, Centro de Biociências, Lagoa Nova s/n, CEP 59078-970, Natal, Rio Grande do Norte, Brazil

a r t i c l e

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Article history: Received 25 April 2011 Received in revised form 1 June 2011 Accepted 4 June 2011 Keywords: Boxfish Ostraciidae Tetraodontiformes Fish cytogenetics Genomic evolution

a b s t r a c t Some groups of fish, such as those belonging to the Order Tetraodontiformes, may differ significantly in the amount and location of heterochromatin in the chromosomes. There is a marked variation in DNA content of more than seven-fold among the families of this Order. However, the karyoevolutionary mechanisms responsible for this variation are essentially unknown. The largest genomic contents are present in species of the family Ostraciidae (2.20–2.60 pg). The present study cytogenetically characterized two species of the family Ostraciidae, Acanthostracion polygonius and A. quadricornis, using conventional staining, C-bandings, Ag-NOR, CMA3/DAPI, AluI, PstI, EcoRI, TaqI and HinfI restriction enzymes (REs) and double FISH with 18S and 5S rDNA probes. The karyotypes of both species showed 2n = 52 acrocentric chromosomes (FN = 52; chromosome arms) and pronounced conserved structural characteristics. A significant heterochromatic content was observed equilocally distributed in pericentromeric position in all the chromosome pairs. This condition is unusual in relation to the karyotypes of other families of Tetraodontiformes and probability is the cause of the higher DNA content in Ostraciidae. Given the role played by repetitive sequences in the genomic reorganization of this Order, it is suggested that the conspicuous heterochromatic blocks, present in the same chromosomal position and with apparently similar composition, may have arisen or undergo evolutionary changes in concert providing clues about the chromosomal mechanisms which led to extensive variation in genomic content of different Tetraodontiformes families. © 2011 Elsevier B.V. All rights reserved.

1. Introduction There are marked differences in DNA content among the families of Tetraodontiformes. This Order includes the family Tetraodontidae, which has the lowest DNA content among vertebrates (0.34–1.00 pg) (Noleto et al. 2009), groups with content similar to that of the Perciformes (1.7 pg), such as the superfamily Balistoidea (1.14– 1.49 pg), and Ostraciidae, which exhibit the largest DNA content per cell, with values ranging from 2.20 to 2.60 pg (Brainerd et al., 2001). The genomic variation observed in this group is presumably due to the evolutionary loss of repetitive DNA or other noncoding DNA (Neafsey and Palumbi 2003). Variations in the number of repetitive sequences, satellite DNA, transposition elements (TE) and the ribosomal genes cause notable differences in genome size (C value) among eukaryotic species (Biemont, 2008). In protozoa, genomic content varies by up to 5800 times, arthropods, 250 times, fish, 350 times, algae, 5000 times and

⁎ Corresponding author at: Departamento de Biologia Celular e Genética, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Brazil. Tel.: + 54 8487285054. E-mail address: [email protected] (P.A. Martinez). 1874-7787/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.margen.2011.06.001

angiosperms, 1000 times (Gregory, 2001). This degree of variation is not related to the biological complexity of the organisms (Petrov, 2001). Constitutive heterochromatin (HC) contributes with a varied parcel of the genome of organisms. In Arabidopsis thaliana it corresponds to around 5%, in Drosophila and humans it reaches 30%, while it accounts for up to 80% of the genome in nematodes (Rossi et al. 2007). HC can be found in any region of the chromosomes, but is generally located near the centromeres and associated to the telomeric regions. It is also found, albeit less frequently, in the interstitial regions of the chromosomes (Corradini et al. 2007; Probst and Almouzni, 2008). In addition to other repetitive sequences, this genome fraction includes transposition elements and satellite DNA, which resides mainly in the pericentromeric regions of the chromosomes (Chaves et al. 2000). In some groups, such as Anostomidae (Characiformes), heterochromatinization processes play an important role in chromosomal evolution (Margarido and Galetti, 2000; Galetti et al., 1991). This plasticity involving heterochromatin regions is also present in closely related species that may differ significantly in the amount and location of heterochromatin in their chromosomes, including sex chromosomes, Bs chromosomes and pairs bearing ribosomal cistrons (Sumner, 2003???; Molina and Galetti, 2007, Martinez et al. 2010). However, in the family Ostraciidae there are no cytogenetical data available on the amount and composition of heterochromatin.

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Cytogenetic data from different banding methods in Ostraciidae (boxfishes), a group whose phylogenetic relationships are not totally explained (Holcroft, 2005) and whose genomic content is high (Brainerd et al., 2001), provide a suitable model to clarify the karyotypic and genomic evolution in Tetraodontiformes. This work performs a detailed cytogenetic characterization of two Atlantic species of Ostraciidae (Acanthostracion polygonius and A. quadricornis), inferring about heterochromatic DNA content features in this peculiar Tetraodontiformes group, using Giemsa staining, C-banding, Ag-NORs, CMA3/DAPI, treatment with AluI, TaqI, HinfI, EcoRI and PstI restriction enzymes and double-FISH with 18S and 5S probes.

2. Materials and methods Karyotype analyses were carried out in 13 specimens collected on the coast of Salvador, Bahia (12 o58′S, 38 o31′W), in Northeast Brazil. These included three of A. polygonius and ten of A. quadricornis. The specimens were submitted to mitotic stimulation for a period of 24–48 h (Molina et al. 2010) and mitotic metaphases were obtained from cell suspensions of the anterior portion of the kidneys, following the in vitro methodology proposed by Gold et al. (1990). The heterochromatic regions were defined according to Sumner (1972), while Ag-NOR detection was based on Howell and Black (1980). Staining with CMA3 and DAPI fluorochromes followed Schweizer's protocol (1980). The chromosome digestion patterns of AluI, EcoRI, PstI, HinfI and TaqI restriction enzymes were determined by tests involving enzymes concentration, exposure time and temperature, as proposed by Cau et al. (1988). As per manufacturer's instructions, a total of 40 μl, at a concentration of 10 U/μl of enzyme diluted in buffer, was placed on the prepared slide, which was covered with a coverslip. The AluI enzyme was incubated for 4–5 h, and the EcoRI, PstI, HinfI and TaqI enzymes for 10–12 h in a moist chamber at 37 °C, except TaqI, which was incubated at 65 °C. The slides were then washed in distilled water and stained with 5% Giemsa diluted in phosphate buffer, pH 6.8 and air dried. The double FISH technique applied was based on the procedure adopted by Pinkel et al. (1986), with a number of modifications aimed at improving preparation quality. The 18S and 5S rDNA probes were obtained by polymerase chain reaction (PCR) amplification of the DNA of Prochilodus argenteus (Hatanaka and Galetti, 2004) and Leporinus obtusidens (Martins and Galetti, 1999), respectively. Fluorescent in situ hybridization (FISH) was performed on mitotic chromosome spreads. The metaphase chromosome slides were incubated with RNAse (40 μg/mL) for 1.5 h at 37 °C. After denaturation of chromosomal DNA in 70% formamide, spreads were incubated in 2 × SSC for 4 min at 70 °C. Hybridization mixtures containing 100 ng of denatured probe, 10 mg/mL dextran sulfate, 2 × SSC, and 50% formamide in a final volume of 30 μl were dropped onto the slides, and hybridization was performed overnight at 37 °C in a moist chamber. Post hybridization washes were carried out at 37 °C in 2 × SSC, 50% formamide for 15 min, followed by a second wash in 2 × SSC for 15 min, and a final wash at room temperature in 4 × SSC for 15 min. The 18S rDNA probes were PCR labeled with biotin-dUTP and the 5S rDNA with digoxigenin-dUTP. Detection of the 18S rDNA probes were obtained with FITC-avidin and the 5S rDNA probes were detected with rhodamine-anti-digoxigenin conjugate. The chromosomal preparations were counterstained with DAPI (0.2 μg/ml). The metaphases were photographed by an Olympus BX50 epifluorescence microscope (Olympus Corporation, Ishikawa, Japan), equipped with a DP70 digital image capture system using DPController 1.2.1.108 software (Olympus Optical Co.). The chromosomes were divided into groups according to the centromere position in metacentrics, submetracentrics, subtelocentrics and acrocentrics and arranged in the karyotype in descending order of size.

3. Results The cytogenetic analyses performed on the Acanthostracion quadricornis and A. polygonius identified a common diploid number of 2n = 52 chromosomes, with the entire complement composed of acrocentric elements (FN = 52). When submitted to the different bandings used here, the chromosomes from both species showed similar structural patterns. The Ag-NOR technique revealed active ribosomal sites in interstitial position in the 13th chromosome pair (Fig1a,b). The karyotypes of the species show all the chromosomes bearing large heterochromatic blocks in pericentromeric position. In addition, interstitial heterochromatin were present in chromosome pair 12 and 13 (CMA3+/DAPI-; coincident with the NORs) and 21 (Fig. 1c,d and Fig. 2a,b). Double fluorescence in situ hybridization allowed the identification of a non-syntenic condition between 18S and 5S ribosomal subunits. The sequences of 18S rDNA subunit were coincident in both A. polygonius and A. quadricornis with Ag-NOR sites in the 13th pair, while the 5S rDNA sites were in interstitial position in the 11th pair (Fig.1e,f). Sequential CMA3/DAPI staining showed that, besides the nucleolar bearing region, the pericentromeric and telomeric regions of some chromosome pairs revealed CMA3+ (Fig.2a,b). Positive signals in the pericentromeric sites were observed from treatments with AluI, TaqI, HinfI and EcoRI restriction enzymes. A slight divergence in the intensity of non-digested bands was also observed (Fig.2c-l). Treatments with the AluI enzyme showed a number of chromosomes with interstitial sites. The TaqI enzyme also highlighted a conspicuous non-digested telomeric region in one of the chromosome pairs. EcoRI produced a pattern of discernible longitudinal bands, while exposure to PstI led to uniform digestion of all the chromosomes, evidencing the presence of cleavage sites equitably distributed in the chromosomes of the two species. Both species showed a similar band pattern arising from digestion by the five restriction enzymes used (Table 1). 4. Discussion Tetraodontiformes families have generally shown extreme karyotypic diversification, in both the numerical and structural karyotype aspects, with diploid values ranging from 2n = 28 to 52 chromosomes. They also exhibit considerable differences in fundamental number (FN), which varied between 33 and 72 (Sá-Gabriel and Molina, 2005). The chromosomal data available for the Order point out to different karyoevolutionary tendencies, with a number of structural rearrangements, such as centric fissions, fusions and especially pericentric inversions occurring preferentially in some families and not in others (Arai and Nagaiwa, 1976; Molina and Galetti, 2007). Whereas considerable cytogenetic information is available for some families, it is practically nonexistent in others. This is particularly so in Ostraciidae, where only five of the 33 species of the family have been analyzed in terms of their chromosomal patterns. The cytogenetic data presented here shows the marked variation in diploid values in this family, although this variation is less in relation to FN. Lactaria diaphana exhibited the lowest diploid value, with 2n = 36 chromosomes (FN = 48), while Ostracion cubicus and O. immaculatus showed 2n = 50 chromosomes (FN = 54) (Arai and Nagaiwa, 1976; Arai, 1983). The chromosome numbers presented here for A. polygonius and A. quadricornis (2n = 52, all acrocentric) represent the highest diploid values described for Ostraciidae, corroborating the wide numerical variation existing in this family. The largest chromosome pairs in the karyotype of A. polygonius and A. quadricornis varied from 2.5 to 4.5 μm, while the largest elements in the karyotype of L. diaphana, with approximately 8.0 μm

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Fig. 1. Karyotypes of A. polygonius (a, c, e) and A. quadricornis (b, d, f) using conventional staining (a, b). Note the nucleolar organizer regions. C-banding (c, d), 18S rDNA (green) and 5S rDNA (red) FISH (e, f). Bar = 5 μm.

(large metacentric chromosomes), were double or triple this size (Arai, 1983). Thus, the numerical chromosomal variation and smaller amplitude in the number of chromosome arms present in Ostraciidae indicate the occurrence of mainly fissions or Robertsonian fusions, complemented by pericentric inversion events in the modeling of the family's karyotype. The nucleolar organizer regions located in interstitial position in the 13th pair, identified by Ag-NORs and 18S rDNA FISH in the karyotypes of Atlantic boxfish A. polygonius and A. quadricornis, suggest that they are homologous pairs. This pattern of single NORs is considered a common character, not only for teleosts, but also for most vertebrate species (Amemiya and Gold, 1986). The association between NORs sites and CMA3+ heterochromatin regions, observed in both species analyzed is often described in fishes (Amemiya and Gold, 1986; Almeida-Toledo et al. 1996), where these regions are adjacent, overlapped or interspersed with the ribosomal sequences, a situation that may favor rearrangements in NOR-bearing chromosomes (Vicari et al. 2003). The 5S ribosomal sites were observed in interstitial position in the 11th pair in both species. The interstitial positioning of these sites has been considered a plesiomorphic character in fish (Pendás et al. 1994; Martins and Galetti, 1999, 2001). It has been suggested that their greater frequency in this position provides protection against disruptive evolutionary events, such as transposition, which generally acts as a dispersive mechanism of the sequences located in terminal positions in the chromosomes (Noleto et al. 2007). However, due to limited chromosomal mapping data of 5S rDNA sites in fish, their purely haphazard occurrence in interstitial position, situation that likely occurs for innumerable other genes, cannot be ruled out. A non-syntenic condition between 5S and 18S rDNA subunits is the most common pattern found in vertebrates (Lucchini et al. 1993; Suzuki et al., 1996; Martins and Galetti, 2001). This was also observed in other species of Tetraodontiformes, such as Chilomycterus spinosus (Diodontidae) and in species of Tetraodontidae, Sphoeroides greeleyi, S. testudineus (Noleto et al. 2007), Tetraodon fluviatilis (Mandrioli et al.

2001) and T. nigroviridis (Fischer et al. 2000). Although data on the position and frequency of ribosomal sites in A. polygonius and A. quadricornis suggests a plesiomorphic condition, there are no other data on Ostraciidae that allow comparisons to be made. Heterochromatic regions are highly variable among organisms (Brutlag, 1980). Thus, they have played a key role in the karyotypic evolution of different fish lineages. Their variation and diversity may be present at different levels, such as intra-population polymorphisms (e.g. Gymnothorax vicinus, Vasconcelos and Molina, 2009), interpopulation polymorphisms, such as in Hoplias malabaricus (Jacobina et al. 2009) and Leporinus elongatus (Molina et al. 2008) or interspecific diversification (Moreira-Filho and Bertollo, 1991). Heterochromatic polymorphisms in fish are generally discrete, involving a number of chromosome pairs, possibly related, among other factors, to the size of heterochromatic blocks (Martínez et al., 1991; Jankun et al. 1995) or as a result of the association between the heterochromatic regions and polymorphic NORs (Hartley, 1988). However, there are cases in which polymorphisms are extensive on a large number of chromosome pairs, such as in A. scabripinnis (Mantovani et al. 2000). In contrast with this evolutionary plasticity, the absence of numerical and position differences as well as in the composition of the heterochromatic portions of the chromosomes of the two species is surprisingly, especially due to the dynamism normally identified in repetitive DNA. In these species, the pericentromeric heterochromatic portions, as well as the telomeric portions of a number of pairs, revealed GC-rich composition. This situation has also been described for other species of fish and vertebrates (Mayr et al. 1986; Fernandez-Garcia et al. 1998; Caputo et al. 2003). Chromosomal similarities between the two karyotypes were highlighted by the responses to treatments with the restriction endonucleases used. Thus, the large heterochromatic blocks were selectively preserved from the action of AluI, TaqI and HinfI enzymes and digested by PstI. However, the interstitial heterochromatin C + observed in pairs 12 and 21 indicate a distinct origin, identified

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Fig. 2. DAPI/CMA3 staining (a, b), treatment with AluI (c, d), TaqI (e, f), HinfI (g, h), EcoRI (i, j) PstI (k, l) restriction enzymes in metaphasic chromosomes of A. polygonius and A. quadricornis respectively. Bar = 5 μm.

for not exhibiting CMA3+ response. These regions, which were intensively digested by TaqI, HinfI and PstI enzymes and mildly by the AluI enzyme, reinforce this hypothesis. The action of EcoRI gave rise to longitudinal bands, a condition also observed in other species of marine fish such as Apogon americanus (Perciformes), in which the pattern of digestion exhibited by the EcoRI enzyme was similar to that of the replication bands (Araujo et al. 2010). The cleavage sites of this enzyme may be related to the functional characteristics of chromosomal replication. The occurrence of a large heterochromatic content with the same compositional pattern allows infer a common origin owing to in concert heterochromatinization events. An important functional role has been attributed to centromeric repetitive sequences (Redi et al. 1990; Garrido-Ramos et al. 1995; Slijepcevic et al., 1997; Canapa et al. 2002), and telomeric sequences

Table 1 Common response patterns of chromosomes from A. polygonius and A. quadricornis to C-banding and digestion with different restriction enzymes. Markings Treatment

Centromeric

Interstitial

Telomeric

Longitudinal

C-banding AluI TaqI HinfI EcoRI PstI

+ + + + + −

+ + − − − −

− − + − − −

− − − − + −

(Garagna et al. 1995; Nanda et al. 1995; Slijepcevic et al. 1997; Canapa et al. 2002), rather than a merely neutral role. Constitutive heterochromatin performs innumerable essential functions, bearing genes that may be related to viability and fertility and is thus a characteristic conserved in the evolution of the eukaryotic genome (Corradini et al. 2007). The repetitive character of the centromeric and telomeric regions may influence the frequency of structural reordering in the surrounding regions, a critical point for inserting or retaining repetitive sequences. These areas show radical alterations in gene reordering and are the preferential sites of reciprocal translocations. Some analyses of the fusion points of chromosomes that undergo Robertsonian translocations (Rb) show that the rupture centers are inside the satellite DNA. It has been proposed that the centromeric DNA-binding protein (CENP-B) facilitates these types of reordering (Kipling and Warburton 1997; Garagna et al., 2001). If the large amount of pericentromeric and telomeric heterochromatin contained in the chromosomes of Ostraciidae is a vestige of Tetraodontiformes ancestry, its concert action must have played an important role through the different chromosomal rearrangements that occurred in the modeling of the current karyotypes of Tetraodontiformes species. Given that the selective pressures on these regions tend to be lower, they may have acted as facilitator sites for different types of rearrangements, such as fusions, fissions and chromosomal inversions, frequent in some families of the Order. The degree of heterochromatin condensation in the centromere regions may promote or prevent fusion events (Slijepcevic et al. 1997), which, in turn, seem to be influenced by GC-rich sequences. AT-rich

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centromeric regions may control DNA curvature (Canapa et al. 2002), and it was observed that greater DNA curvature promotes faster chromatin spiralization (Radic et al. 1987), which reduces the possibility of centric fusions (Garrido-Ramos et al. 1995). On the other hand, GCrich regions have a tendency for high recombination rates and could favor centric fusions or fissions (Redi et al. 1990). This seems to be the case in the species of Acanthostracion analyzed. 5. Conclusions Our data, showing the similar location and composition of the heterochromatic portion between the karyotypes of the two species of Acanthostracion, suggest a common origin, and not the result of convergent evolution that led to a mere increase in repetitive DNA in these species. These regions, acting in concert, might be responsible for the marked differences of genomic size of Tetraodontiformes groups. Acknowledgements The authors wish to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – Proc. 556793/2009-9). We also thank José Garcia Jr. for species identification and Francisco Rivera for suggestions on the manuscript. References Almeida-Toledo, L.F., Stocker, A.J., Foresti, F., Almeida Toledo-Filho, S., 1996. Fluorescence in situ hybridization with rDNA probes on chromosomes of two nucleolus organizer region phenotypes of a species of Eigenmannia (Pisces, Gymnotoidei, Sternopygidae. Chromosome Res. 4, 301–305. Amemiya, C.T., Gold, J.R., 1986. Chromomycin A3 stains nucleolus organizer regions o fish chromosome. Copeia 226–231. Arai, R., Nagaiwa, K., 1976. Chromosomes of Tetraodontiform fishes from Japan. Bull. Natn. Sci. Mu. 2, 59–72. Arai, R., 1983. Karyological and osteological approach to phylogenetic systematic of tetraodontiform fishes. Bull. Natn. Sci. Mus. 9, 175–210. Araujo, W.C., Martinez, P.A., Molina, W.F., 2010. Mapping of ribosomal DNA by FISH, EcoRI digestion and replication bands in the cardinalfish Apogon americanus (Perciformes). Cytologia 75 (1), 109–117. Biemont, C., 2008. Within-species variation in genome size. Heredity 101, 297–298. Brainerd, E.L., Slutz, S.S., Hall, E.K., Phillis, R.W., 2001. Patterns of genome size evolution in tetraodontiform fishes. Evolution 55, 2363–2368. Brutlag, D.L., 1980. Molecular arrangement and evolution of heterochromatic DNA. Ann. Rev. Genet. 14, 121–144. Canapa, A., Cerioni, N., Barucca, M., Olmo, E., Caputo, V., 2002. A centromeric satellite DNA may be involved in heterochromatin compactness in gobiid fishes. Chromosome Res. 10, 297–304. Caputo, V., Colomba, M., Nisi Cerioni, P., Viturri, R., Giovannotti, M., Olmo, E., 2003. Chromosome banding and molecular cytogenetic study of two Mediterranean trachinoid fish species (Teleostei: Trachinidae, Uranoscopidae). Cytogenet. Genome. Res. 103, 139–143. Cau, A., Salvadori, S., Deiana, A.M., Bella, J.L., Mezzanotte, R., 1988. The characterization of Muraena helena L. mitotic chromosomes: karyotype, C-banding, nucleolar organizer regions and in situ digestion with restriction endonucleases. Cytogenet. Cell. Genet. 47, 223–226. Chaves, R., Guedes-Pinto, H., Heslop-Harrison, J., Schwarzacher, T., 2000. The species and chromosomal distribution of the centromeric alpha-satellite I sequence from sheep in the tribe Caprini and other Bovidae. Cytogenet. Cell. Genet. 91, 62–66. Corradini, N., Rossi, F., Giordano, E., Caizzi, R., Vern, F., Dimitri, P., 2007. Drosophila melanogaster as a model for studying protein-encoding genes that are resident in constitutive heterochromatin. Heredity 98, 3–12. Fernandez-Garcia, J.L., Martínez-Trancón, M., Rabasco, A., Padilla, J.A., 1998. Characterization of the heterochromatic chromosome regions in sheep. Genes. Genet. Syst. 73, 45–50. Fischer, C., Ozouf-Costaz, C., Crollius, H.R., Dasilva, C., Jaillon, O., Bouneau, L., et al., 2000. Karyotype and chromosomal location of characteristic tandem repeats in the pufferfish Tetraodon nigroviridis. Cytogenet. Cell. Genet. 88, 50–55. Galetti Jr., P.M., Mestriner, C.A., Venere, P.C., Foresti, F., 1991. Heterochromatin and karyotypic reorganization in fish of family Anostomidae (Characiformes). Cytogenet. Cell. Genet. 56, 116–121. Garagna, S., Broccoli, D., Redi, C.A., Searle, J.B., Cooke, H.J., Capanna, E., 1995. Robertsonian metacentrics of the house mouse lose telomeric sequences but retain some minor satellite DNA in the pericentromeric area. Chromosoma 103, 685–692. Garagna, S., Marziliano, N., Zuccotti, M., Searle, J.B., Capanna, E., Redi, C.A., 2001. Pericentromeric organization at the fusion point of mouse Robertsonian translocation chromosomes. Proc. Natl. Acad. Sci. USA 98, 171–175.

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Msc. Pablo Ariel Martinez Master in Aquatic Ecology. Developed his thesis on molecular cytogenetics of Tetraodontiformes. Currently is attending a doctorate in Ecology in the Universidade Federal do Rio Grande do Norte (Natal, Brazil). Has experience in molecular cytogenetics and geometric morphometry of vertebrates. Msc. Uedson Pereira Jacobina Master in Zoology. Currently is attending a doctorate in Biotechnology in the Universidade Federal do Rio Grande do Norte (Natal, Brazil). Has conducted research in molecular cytogenetics of freshwater and marine fish; with experience in FISH, fluorochrome and replication banding. Ph.D. Wagner Franco Molina Doctor in Genetics and Evolution. Has experience in animal genetics, with emphasis on animal cytogenetics. Developed research mainly on marine fish cytogenetics and improving of white shrimp Litopenaeus vannamei through chromosomal manipulation and DNA markers.