Chromosome preparation in fish: effects of fish species and larval age

Abstract

To date, several protocols have been developed to achieve clear and identifiable metaphase chromosome spreads from larvae of a single fish species. However, the efficiency of these protocols in more than one fish species has barely been compared within a single study. This work investigated the dependency of chromosome preparation parameters including colchicine concentration (0.01, 0.025, 0.05 %) and exposure duration (3, 5 h), hypotonic solution (distilled water, 0.075 M KCl solution), and Giemsa stain solution concentration (6, 8, 10, 11, 12, and 14 %) and incubation period (15, 30, 45, and 60 min) to two species of fish, the African catfish (Clarias gariepinus) and the zebrafish (Danio rerio) at different larval ages (0, 2, and 4 days post-hatch, dph). Results indicated that larval age, colchicine concentration and/or incubation time, and/or the type of hypotonic solution varied with fish species while staining the chromosomes with 11 % Giemsa solution for 45 min can be maintained regardless of the species or larval age. Interestingly, employing the selected values from diploid C. gariepinus experiment to prepare metaphase chromosomes from larvae of their triploid siblings proved to be efficient.

Peer Review #1 of "Optimization of the cytogenetic protocol for Pangasianodon hypophthalmus (Sauvage, 1878) and Clarias gariepinus (Burchell, 1822) (v0.1)"

“…No alternative fixative, ratio or duration of exposure was considered in this study as freshly prepared Carnoy's solution (methanol-acetic acid at ratio 3:1) is commonly used with a unanimous exposure time of 20 mins with three changes (Fopp-Bayat and Woznicki 2006; Karami et al, 2010;Pradeep et al, 2011 etc.). However, the best time for making cell suspension was determined by initiating the process (i.e.…”

“…Several techniques have been optimized to obtain well spread mitotic chromosomes in fish and this may differ for different species (e.g. as observed by Karami et al, 2010). These are usually in terms of chemical types, concentration, and duration of exposure.…”

“…Despite the ease of chromosome preparation from eggs and larvae of fish (Shao et al, 2010, Karami et al, 2010Amar-Basulto et al, 2011;Botwright 2015), cytogenetic studies involving specific tissues of fingerlings, juvenile or adult fish may have some merits over the use of whole larvae or egg. Aside the possibility of having a large amount of tissue from which metaphase chromosome can be isolated multiple times; this method has a pride of place in the characterization of the progenies of intergeneric hybridization (distance crosses) between different fish species from different genus (Okomoda et al, 2018).…”

Changes in nutritional parameters in diploid and triploid African catfish Clarias gariepinus following chlorpyrifos exposure

“…Each batch of eggs was transferred to a 2000 l fiberglass tank filled with 500 l UV-sterilized water. To confirm successful initial triploidy induction, the chromosomal spreads of 20 larvae from the cold shock treated groups were prepared according to the method of Karami et al (2015a). Larvae were initially fed ad libitum with freshly hatched Artemia nauplii 3 times a day for 1 wk.…”

Comparison of different ploidy detection methods in Oncorhynchus mykiss, the rainbow trout

“…Chromosome preparation is known to be very random (Deng et al 2003). There are too many factors to consider such as relative humidity (Spurbeck et al 1996), cell dropping height (Barch et al 1997;Hlics et al 1997), and flame vs. air drying method of the slide (Karami et al 2015). Still, even taking into account all these aspects, getting a well-spread metaphase is overly time consuming and not always rewarding.…”