Introduction

The family Batrachoididae is a small group of fish, commonly known as toadfish, containing around 70 species divided into 21 genera and three subfamilies (Nelson 1994; Greenfield et al. 1994; Collette 1995, 2001; Greenfield 1997). Although no fishing statistics are available, larger species of toadfishes are commonly found in local markets and may fetch high prices in Venezuela and French Guiana (http://www.fishbase.org/Summary/FamilySummary.cfm?ID=189).

The Batrachoididae family includes four species (Opsanus tau, O. beta, Porichthys notatus and Halobatrachus didactylus) which are considered important experimental organisms for biomedical and physiological studies (see Freshwater et al. 2000 and Palazón-Fernández et al. 2001 for references).

Cytogenetic data on batrachoidid species are restricted to number and chromosome formulae for Amphychthys cryptocentrus, Batrachoides manglae and Thalassophryne maculosa from Venezuela (Nirchio et al. 2002b, 2004), B. pacifici from Panama (Nirchio et al. 2002a), Porichthys porosissimus from Brazil (Brum et al. 2001) and H. didactylus from Spain (Palazón et al. 2003).

In this study we describe the diploid number, chromosome formulae, C-band pattern, and NOR locations in Porichthys plectrodon from Margarita Island, Venezuela. This toadfish is found in the western Atlantic Ocean from Virginia to Brazil (Hoese and Moore 1998) and is one of the five species of toadfish recognized in Venezuela (Cervigón 1993). This species is found on sand or mud beds, mostly at depths between 10 and 250 m, in a wide variety of salinities and temperatures (Lane 1967; Moore 1970).

Materials and methods

Eleven specimens (eight males, three females) of P. plectrodon from Margarita Island, Venezuela were analyzed. Voucher specimens were deposited at the Ichthyology Collection of the Escuela de Ciencias Aplicadas del Mar, Universidad de Oriente (Venezuela).

The preparation of chromosomes was performed according to Nirchio et al. (2002b) but reducing the colchicine incubation time. Each specimen was injected intraperitoneally with a colchicine solution (0.5%; 1 ml/100 g fish weight). The fish were maintained in a well-aerated aquarium and after 3 h they were sacrificed, and the kidneys were extracted and placed in a hypotonic solution of 0.4% KCl. Every single kidney was minced with fine forceps and then with a glass syringe by repeated aspiration and forced release until a fine cellular suspension was obtained. After 30 min in the hypotonic solution, the cellular suspension was centrifuged at 1,000 rpm for 3 min. The hypotonic solution was discarded and the cellular button was suspended and washed three times in a methanol-acetic acid mixture 3:1 (V:V).

One droplet of the cellular suspension was dropped on a clean microscope slide, previously chilled in a freezer, from a height of 45 cm. The slides were briefly put over a flame and then allowed to air-dry.

For the conventional karyotype, the preparations were stained for 20 min with 5% Giemsa in phosphate buffer pH 6.88. Detection of the nucleolus organizer regions (NORs) was done following the silver staining method of Howell and Black (1980). C-bands were obtained according to the methods described by Sumner (1972).

A total of 134 mitotic spreads (10–12 plates per individual) were scanned to determine the modal chromosome number, and 55 spreads (five plates per individual) were used to determine the number of NORs.

The mitotic figures were photographed using a green filter and 50 ASA film. The resulting photographs were then scanned and stored as *.tif images. Long arm (L), short arm (S) and whole chromosome length were measured for each chromosome to the nearest 0.01 mm, using the measuring tool in Adobe Photoshop software v.7.0. The length of each chromosome pair in relation to the total chromosome length (RL%) was obtained from these values. Chromosomes were identified by the arm ratio criteria proposed by Levan et al. (1964).

Results

All specimens of P. plectrodon under study were characterized by a diploid chromosome count of 2n=44, obtained in 97% of cells examined (130 cells). The hypomodal and hypermodal counts, on the whole, barely reached 3% of all the cells recorded, and probably resulted from preparation-caused defects such as chromosome loss, overlap, miscounting, and additional chromosomes from another spread.

The standard karyotype of the species, prepared by arrangement of chromosomes into groups based on L/S ratio, is shown in Fig. 1. No differences were observed between sexes. This karyotype was composed of 8 metacentric (M), 10 submetacentric (SM), 6 subtelocentric (ST), and 20 acrocentric (A) elements, with an arm number (NF=Nombre Fondamentale) of 62. The length of the M chromosomes compared to the total length of the diploid chromosome length (RL%) was 29.5%, ranging from 3.1% to 13.5% per chromosome. For the SM chromosomes, RL% was 19.7%, ranging from 3.2% to 4.9%. For the ST chromosomes, RL% was 12.5%, ranging from 3.9% to 4.4%. For the A chromosomes, RL% was 38.3%, ranging from 2.3% to 4.5%. The sizes of the metacentric chromosomes were very distinct: each pair, from the smallest to the largest, was approximately half the size of the following pair, making the identification of homologous pairs a simple task. However, minimal differences in chromosome size and arm ratio in the submetacentric, subtelocentric and acrocentric chromosomes made it difficult to classify homologous pairs with such certainty. Table 1 summarizes chromosome measurements of the different pairs.

Fig. 1 a
figure 1

Karyotype of Porichthys plectrodon stained conventionally with Giemsa. NOR-bearing chromosomes appear in the square inset. b C-banded karyotype. Bar 10μm

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Table 1 Chromosome number and formulae in six species of toadfish. NF Arm number (Nombre Fondamentale), M metacentric, SM submetacentric, ST subtelocentric, A acrocentric
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Silver NOR staining of the selected metaphase spreads revealed one pair of small-sized acrocentric NOR-bearing chromosomes with black dots located terminally on the short arms of these chromosomes. They could be identified as pair 21 (Fig. 1A). No NOR polymorphisms were observed, but in several cases an intimate association was seen between the NOR-bearing arms of the two homologous chromosomes (Fig. 2C).

Fig. 2a–c
figure 2

Metaphase plates of Porichthys plectrodon stained for NOR. Observe constancy of NOR (a, b) and NOR-bearing chromosome association (c)

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The visualization of C-banding revealed large positive pericentromeric bands at pairs 1 and 2. The heterochromatic blocks occupy approximately 38% of the chromosomes in pair 1 and around 27% in pair 2. In the rest of the chromosomes, heterochromatin was found at centromeric positions, some more conspicuous than others (Fig. 1B).

Discussion

Available data on the karyotype of batrachoidid species report a diploid chromosome number of 2n=46, a condition repeated in several species, with the exception of P. porosissimus (Brum et al. 2001) and P. plectrodon, which each possess a complement of 2n=44 (Table 2). Assuming that the karyotype for present teleosteans derives from an ancestral diploid number of 2n=48 uniarmed chromosomes (Ohno 1974; Gold 1979; Vitturi et al. 1995), a chromosome number under 2n=48 (Table 2) and the presence of a high number of biarmed elements could indicate that the karyotype of P. plectrodon is a derivative character (apomorphic) among Batrachoididae.

Table 2 Mean±SD of the absolute length of each chromosome pair [AL(µm)], length of each chromosome as percentage of total length of the haploid complement (RL%), value of the arm ratio (C) and type of chromosome morphology according to arm ratio (T)
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C-banding revealed that the heterochromatin in P. plectrodon occupies a wide pericentromeric region of the pairs 1 and 2. Other less conspicuous pericentromeric bands were observed in most of the chromosomes (Fig. 1B). This pattern of heterochromatin distribution is similar to the one reported for P. porosissimus (Brum et al. 2001) and can be attributed to the Robertsonian fusion of two large pairs of uniarmed chromosomes, reinforcing the hypothesis of Brum et al. (2001) regarding the origin of these metacentric chromosome pairs. On the other hand, C-banding in T. maculosa has shown the presence of heterochromatic regions restricted to centromeres (Nirchio et al. 2004). This dissimilar feature between Talasophryne and Porichthys suggests that C-bands could be a good cytotaxonomic marker in the family Batrachoididae.

As far as the available information allows generalization, NORs in the Batrachoididae seem to be a non-conservative feature. Accordingly, P. plectrodon exhibited positive signals on the telomeric regions of the short arm of pair 21 (Fig. 1A); Halobatrachus didactylus possesses a single pair of NOR-bearing chromosomes but submetacentric instead of subtelocentric (Palazón et al. 2003). Unsatisfactory results were obtained by Brum et al. (2001) when attempting to detect NORs by silver impregnation in P. porosissimus, but they inferred the occurrence of more than a single NOR-bearing pair on the basis of the presence of one to three nucleoli per nucleus, whereas Nirchio et al. (2004) established that T. maculosa does not possess additional NORs since fluorescence in situ hybridization revealed fluorescent signals on the telomeric region of the short arm of only one medium-sized subtelocentric chromosome pair (pair 16), the same that was identified using silver salts.

Although more information is required for inferring phylogenetic relationships between batrachoidid species, available data suggest that chromosome translocation events involving the active NOR sites detectable by silver impregnation seem to be associated with the diversification in the group; hence NOR sites have a potentially high cytotaxonomical value within this fish group.

An interesting feature observed in this study was an intimate association between the NOR-bearing arms of two homologous acrocentric chromosomes (Fig. 2C). Similar behaviour has occasionally been observed in Gobius fallax (Thode et al. 1983) and Cyprinus carpio (Anjum and Jankun 1998). A study using several staining techniques in Oedalechilus labeo (Rossi et al. 2000) has also presented two active NOR-bearing chromosomes associated at the tips of their NOR-bearing arms, but their association pattern was not described. According to Anjum and Jankun (1998), such associations between chromosomes may result from a tendency of the NOR-bearing chromosomes to be found and most likely reflect their joint participation in the formation of the common nucleolus during the preceding interphase.

For comparative purpose, we calculate the arm number (NF) of all toadfishes so far karyotyped by assigning a value of 2 to biarmed chromosomes (metacentric and submetacentric) and a value of 1 to uniarmed chromosomes (subtelocentric and acrocentric). Thus, NF is 52 for A. cryptocentrus, 58 for B. manglae, B. pacifici and P. porosissimus, 64 for T. maculosa, 66 for H. didactylus and 62 for P. plectrodon (see Table 2 for references). According to LeGrande (1981), differences in the NF among closely related species can be the result of pericentric inversions, whereas the differences in the diploid number (2n) presumably represent Robertsonian changes (fusions, fissions). Thus, the karyotype of P. plectrodon can be interpreted as the result of structural chromosomic rearrangements involving at least the central fusion of four pairs of uniarmed chromosomes to form two large pairs of biarmed elements (metacentric or submetacentric) resulting in the reduction of the diploid chromosome number from the ancestral fish karyotype (2n=48) to 2n=44, as well as a series of pericentric inversions, generating biarmed chromosomes and so increasing the NF.

In conclusion, the chromosome analysis of species within the Batrachoididae family, using conventional staining procedures, reveals a great variability in the size, shape and number of chromosomes, as well as in NOR sites and constitutive heterochromatin distribution, suggesting that the diversification in the family is closely related to numerical and structural changes in the chromosomes. Therefore, cytological information may be a powerful tool in further taxonomical research work on Batrachoididae.