In India, Pandey and Lakra¹⁵ recorded tetraploidy in a single individual of Clarias batrachus, using karyotype (2n = 50; 4n = 100) as the evidence. While listing chromosome number of several fishes, Manna¹⁶ doubted the possible occurrence of polyploidy in Heteropneustes fossilis, but provided no supporting evidence for it. Tiwary et al.¹⁷ claimed successful induction of triploidy in H. fossilis, but have not provided acceptable evidence for their claim. To the best of our knowledge, no report is as yet available for natural occurrence of haploids (male and female) in any fish species, although the publication of Varadaraj¹⁸ stands out as a single publication on the induction of viable haploid gynogenetic Oreochromis mossambicus.

This present communication reports on the natural occurrence of monoploids and polyploids (triploids and tetraploids), both in males and females in the South Indian populations of the catfish, H. fossilis, on the basis of the evidences of erythrocyte nuclear volume and karyotype. We have also shown the unconventional mechanism of spermatogenesis in these naturally occurring polyploids.

Collections of H. fossilis were made from different sources in Tamilnadu and Kerala during April–October 1998. As many as 120 individuals were randomly selected from these populations, for confirmation of their ploidy groups. Initially, a number of individuals were sacrificed to acquire blood for erythrocyte nuclear measurement¹⁹, and tissues for karyotyping²⁰. However, all subsequent analyses were made following noninvasive procedures, as live monoploids and polyploids were required for further studies: Hence, blood was collected by caudal puncturing. Smears were fixed in methanol for 1 min and stained in 4% Giemsa in phosphate buffer (pH 6.4) for 10 min, and were subjected to nuclear measurements using stage and ocular micrometer (Erica, Japan) under a phase contrast microscope (Nikon,
Japan).

Table 1 shows the occurrence of monoploids, diploids, triploids, and tetraploids at the frequency of 1.7, 91.7, 4.2 and 2.5% respectively. In all these unusual ploidy groups, both males and females were recorded, though the frequency of female triploid and tetraploid was only one each, against 4 and 2 males, respectively.

Ploidy was identified on the basis of both erythrocyte (RBC) volume, and chromosome number. The nuclear volume of RBC increased from 4.1 µm³ in a haploid to 8.7, 13.7 and 19.5 µm³ in diploid, triploid and tetraploid, respectively (Figure 1). In tetraploids, the nuclear volume widely varied, compared to other ploidy groups.

The diploid chromosome number varied between 56 and 58. Of the 110 individuals analysed, as many as 86 individuals had 58 chromosomes, 15 individuals had 56, and the rest 57. Of the 120 individuals analysed, 2 proved to be haploids, of which the female bore 30

Figure 1.  Erythrocytes of haploids, diploids, triploids and tetraploid Indian catfish, H. fossilis. All photomicrographs were made at the same magnification of 40 ´ . Note the increase in diameter of the darkly stained nucleus in the haploids, diploid and polyploids.

chromosomes and the male 29 only. All the 5 identified as triploids consistently had 87 chromosomes. No difference was noted in the chromosome number between male and female triploids. The tetraploid carried 116 chromosomes. The presence of microchromosomes were observed in all these ploidy groups (Figure 2).

Table 2 shows the head diameter of euploid and aneuploid sperms generated by haploids, diploids, and polyploids of H. fossilis. In diploids, spermatogenesis is typical and conventional, resulting in the production of 94% euploid (n) sperms; likewise, spermatogenesis was also typical and conventional in tetraploids, resulting in the production of 90% euploid (2n) sperms. But, the spermatogenesis in triploids was unconventional and hazardous resulting the production of 56% euploid sperms and 43% aneuploid sperms. Among the euploid sperms, some were haploids while others were deploids, triploids, and some even tetraploids (Figure 3). The hazardous nature of spermatogenesis is indicated by the presence of hypohaploids, hypodiploids, hypotriploids and hypertriploids. The ability of triploid and tetraploid sperms to activate/fertilize diploid H. fossilis eggs (n) has been reported elsewhere^21,22.

To the best of our knowledge this is the first report on the natural occurrence of male and female haploids in fish; triploids and tetraploids have already been reported, mostly from China, Japan, Europe, and North America¹. Based on a single individual of Clarias batrachus, natural occurrence of tetraploidy in India was claimed¹⁵.

In India, the presence of the genus Heteropneustes is predominently represented by a single species H. fossilis. In recent years the occurrence of H. microps, which hails from Sri Lanka, has been reported (S. Sridhar and M. A. Haniffa, pers. commun.). The morphological character by which these two species are distinguished is by the anal fin, which remains confluent with caudal fin in H. microps, but separate in H. fossilis. That all the 120 individuals analysed in the present work had anal and caudal fins, typical of H. fossilis clearly indicates that the individuals were not hybrids of these

Figure 2.  Karyotypes of the male and female haploids, diploid, triploid, and tetraploid H. fossilis. Photomicrographs for haploids, diploid and triploid were made at the magnification of 100 ´ and for tetraploid at 40 ´ . Arrows indicate the microchromosome(s).

two species. Moreover, the fact that almost conventional but partially hazardous spermatogenesis occurs in tetraploid individuals also confirms that these analysed monoploids and polyploids were neither a new species nor a hybrid.

It is an established fact that whereas triploid females are sterile, males are partially fertile and produce viable sperms¹. However, fertile female triploids have been reported in a couple of carp species^23–25; in these, the pathways through which fertile oocytes are produced differ in general from the conventional one¹. The hazardous pathways through which sperms are generated in these partially fertile triploid males have been broadly indicated by a couple of previous authors from their flow cytometric and cytological studies^26,27. In triploid Pagrus major Kawamura et al.²⁸ reported the production of sperm carrying haploid to heptaploid genome. In the present study too, sperms with haploid to tetra-ploid genome were recorded. It is not clear how from a triploid, sperms carrying 4n–7n genome can be generated.

ACKNOWLEDGEMENTS.  Financial support by the Indian Council of Agricultural Research and the Department of Biotechnology,
New Delhi, is gratefully acknowledged. We are thankful to Dr M. A. Haniffa and Mr S. Sridhar, Palayankottai for their help.