The karyotype of Lates stappersi (Teleostei: Centripomidae): Preliminary results for a Lake Tanganyika endemic predator

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Joan Hardy
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1 The karyotype of Lates stappersi (Teleostei: Centripomidae): Preliminary results for a Lake Tanganyika endemic predator Student: Silas Majambere Mentor: Ellinor Michel Introduction Lake Tanganyika is important not only for its very high biodiversity, but also for the large stock of fish that are of great economic importance for the people around the lake. The pelagic fish catch is comprised of endemic sardine-like clupeids and their endemic centropomid predators. These species are a major resource, providing 60% of the animal protein for residents of the Lake Tanganyika drainage (Coulter 1991). The four predatory centropomid species are all endemics in the genus Lates. Other species of Lates are found in many rivers and lakes of tropical Africa. For example, the Nile perch, Lates niloticus, has been in the headlines in recent years for its impact in Lake Victoria. Endemic Lates species exist in Lakes Albert and Turkana. However, the four Tanganyikan Lates species (L. angustifrons, L. mariae, L. microlepis, and L. stappersi) have a higher level of morphological differentiation than among the other Lates (Coulter 1991). It is possible that they evolved in sympatry in Lake Tanganyika, as the lake has been the cradle for evolution of many endemic species in other taxa. My objective was to do the first karyological study of this genus. This is an staining of the chromosomes so that their number and banding patterns can be determined. The most dramatic results might have shown polyploidy, which can indicate the mechanism of speciation separating these species. However, a detailed study of the chromosomes can also indicate how much these species are sexually differentiated, if there is a possibility of hybridization, and their level of evolutionary divergence. I was limited by both time and specimen availability and thus present results only on one species, L. stappersi. As this is the first ever karylogical results on a Lates species (L. Kuusipalo, L. Hauser, pers.comm.), I hope it provides a basis for and stimulates further research. Material and methods My study had two components, one on the field and the other in the laboratory. I collected samples of L. stappersi during four night fishing trips on Lake Tanganyika. We caught the fish in lift nets and then I dissected the live fish immediately to get fresh gonad tissue. I used gonads because this tissue shows the two important kinds of cell division (mitosis and meiosis) while other tissues show mitosis only. Mitosis results in cells with the diploid number (2n) of chromosomes and meiosis results in cells with the haploid number (n) of chromosomes. Counting the number of chromosomes is much easier with haploid cells, and with the haploid numbers in hand, one can better determine diploid numbers. Each specimen (one gonad from each fish) was divided into seven subsamples to optimize the reactions, as we had to develop our own protocol. I used the protocol proposed by Katja Hora for the study of the karyotype of snails. I took a fresh part of the gonad after dissection and put it in colchicine in distilled water 0.05 %. The colchicine stops the cell division at the stage of metaphase by preventing the formation of microtubules which normally lead the chromosomes to the pole during the anaphase. Metaphase is better for observation of chromosomes because at that stage of cell division chromosomes are condensed and are still grouped together. One of the samples stayed in colchicine for one hour (slide 1) and the others 30 minutes (slide 3, 5, 6 and 7). After the colchicine reaction, I transferred the gonad to Carnoy's fixative. This prevents the desintegration of the cell, keeping the membranes intact, and the DNA is also condensed because of the acid in the

2 Fig. 1: Meiotic Chromosome Counts (n) Frequency Number of Chromosomes

3 fixative. The standard recipe for this fixative is methanol and acetic acid, 3:1, however ethanol can be used instead of methanol and this was the case in my work. The sample can stay in the fixative for a long while I transferred other subsamples of the gonad directly in the fixative without passing in colchicine (slides 2 and 4). This would show whether the colchicine is indispensable to visualize cells in metaphase stage. I then tried to remove all the impurities on the gonad and its membrane. I transferred the gonad in a drop of 50% acetic acid and macerated it with tungsten needles until the solution was homogenized. The advantage of these needles is that the tissue can't stick to them. The acid separates the cells and condenses chromosomes. I cleaned the slide and put on it a drop of the homogenized tissue solution. I let it air dry or put it on a hot plate which contracts the solution progressively while evaporating the acetic acid, leaving the slide clean and easy to observe. When the slide was completely dry, I stained it with a drop of Orcein 2.5% in acetic acid and lactic acid (1:1). Orcein stains the DNA red. After covering the preparation with a cover slip, I dabbed the excess orcein, careful not to move the cover slip, as this would damage the preparation. I let the slide dry for about 10 to 15 minutes. I sealed the slide with nail polish to prevent air from spreading under the cover slip. I then counted the number of chromosomes visible in the best cells, using the Olympus microscope with phase contrast and three objectives; the 40x were used for cell observations and the 100x for counting. I counted chromosomes both in mitotic (2n) and meiotic (n) cells. As the chromosomes are not always optimally spread, or the contents of several nuclei may be mixed, the counts can vary from instance to instance. Thus the data is a repeated sampling arriving at a mean number. The slides can now be made into permanent mounts if remounted in DPX mounting fluid. Results and discussion Table 1 shows the number of chromosomes and how many times the same number was found for each slide (there were 7 different specimens of L. stappersi). Chromosomes were more visible in samples that had been fixed in colchicine versus those that were placed directly in fixative (this is a qualitative result not shown in Table 1). Slides that remained in colchicine 30 versus 60 minutes did not have significantly different chromosome differentiation. Chromosome squashes can vary for several reasons. Counts may be low if a cell is destroyed and its chromosomes separated and spread in different directions. This can explain why I found very low numbers such as 12, 11 or 9 chromosomes in a few cases. Another reason for low counts is that for the same tissue, cells can be in different stages of meiotic cell division, some in leptotene or pachytene stage. In these cases, all the chromosomes are not completely condensed, and as a result are not always well separated visible under microscope. On the other hand, counts may be high if the chromosomes from neighboring broken cells are layered over complete cells. Thus we searched for median numbers indicating the actual haploid and diploid numbers. For some areas of different slides, I could clearly see chromosomes and count them. The number was betweeen 21 and 27 most of the time, with a high frequency on 23 chromosomes (Fig. 1). In other areas the number of chromosomes averaged around 46 chromosomes (Fig. 2). These two observations led me to conclude that the cells with around 23 chromosomes were at the stage of meiosis (haploid, or n chromosomes) and those with around 46 chromosomes were at the mitosis stage (diploid, or 2n chromosomes). However, 31 chromosomes appeared three times in several different areas for one slide, which is difficult to explain. On another preparation I counted between 84 and 90 chromosomes, which may indicate that that the cell was polyploid.

4 Table 1: Number of chromosomes per slide Number Slide 1 Slide 2 Slide 3 Slide 4 Slide 5 Slide 6 Slide 7 Total Total Counts/ slide

5 Fig. 2: Mitotic Chromosome Counts (2n) Frequency Number of Chromosomes

6 Conclusion and recommendations The methodology, which was used by K. Hora for Lavigeria snails, gave good results in this study with Lates fishes. However, a complete study of the karyotype of Lates stappersi can't be completed in a short time of three weeks. I was tempted to conclude that the karyotype of L. stappersii is 23 chromosomes but it is very early to make such conclusion. Some mistakes could have happened especially in counting but the results seem to respect the logic of the two stages of cell division (meiosis and mitosis). This work should be improved to make a definite conclusion about the karyotype not only for this species but also for all the genus and why not for all known fishes of Lake Tanganyika. Acknowledgements Thanks to all the team of Nyanza Project 2000 specially my Mentor Dr. Ellinor Michel. My deep gratitude to Katja Hora for teaching me the methodology and helping me during all the work. References Coulter, G.W Lake Tanganyika and its life. Oxford Univ. Press, London

Meiosis. Bởi: OpenStaxCollege

Meiosis. Bởi: OpenStaxCollege Meiosis Bởi: OpenStaxCollege Sexual reproduction requires fertilization, a union of two cells from two individual organisms. If those two cells each contain one set of chromosomes, then the resulting cell