Abstract
Information about ploidy is important in both commercial and conservation aquaculture and fish research. Unfortunately, methods for its determination, such as karyology, determination of the amount of DNA in a cell using microdensitometry or flow cytometry and/or measuring erythrocytes in a blood smear can be stressful or even destructive. Some of these methods are also limited by the relatively large minimum size of the individual being measured. The aim of this study was to test a new low-stress method of determining ploidy by measuring the size of erythrocytes in the capillaries of a fish, including small individuals. First, we examined diploid and triploid loach (Cobitis sp.) and gibel carp, Carassius gibelio (Bloch, 1782), using flow cytometry and blood smears, with these results being used as a control. Subsequently, we measured the size of erythrocytes in the caudal fin capillaries of anesthetized fishes of known ploidy under a light microscope. For both the loaches and gibel carp, direct observation of the mean erythrocyte size in epithelial fin capillaries provided a consistent and reliable determination of ploidy when compared with the controls based on flow cytometry and blood smears. This new method allows for rapid determination of ploidy in living small fish, where collection of tissue using other methods may cause excessive stress or damage. The method outlined here simply requires the measurement of erythrocytes directly in the bloodstream of a live fish, thereby making it possible to determine ploidy without the need for blood sampling. The method described is sufficiently efficient, less demanding on equipment than many other procedures, can be used by relatively inexperienced personnel and has benefits as regards animal welfare, which is especially important for fish production facilities or when dealing with rare or endangered species.
Highlights
Polyploidy, the multiplication of whole sets of chromosomes beyond the normal set of two, occurs independently in many groups of fish, from sharks to the higher teleosts
Ploidy in these individuals was initially determined by flow cytometry on a blood sample (loach 2n = 103.6% (96.0–112.0); 3n = 153.8% (142.0–164.4); gibel carp 2n = 97.8% (94.4–104.4); 3n = (154.0% (146.6–162.8)) (Flajšhans et al 2005), using the gold fish (Carassius auratus) as a standard (=100%), and a separate blood smear (Boroń 1994) for measurement of length erythrocyte
The fish used for measurement of erythrocyte size in caudal fin capillaries were immobilized on the mechanical stage of an Olympus BX50 light microscope using a 36 × 125 mm ‘pad’ with two overlapping tiles glued to the underlying glass (Fig. 1)
Summary
Polyploidy, the multiplication of whole sets of chromosomes beyond the normal set of two, occurs independently in many groups of fish, from sharks to the higher teleosts. While there are several ways that a polyploid fish can develop, environmental change and hybrid stabilization may play a large role in the initiation of a new polyploid species. Polyploid fish could gain an advantage over diploid fish through increased heterozygosity, the divergence of duplicate genes, and/or increased expression of key physiological proteins (Leggatt and Iwama 2003). Hybridization and polyploidization became increasingly appreciated as important evolutionary mechanisms that even had a profound impact on mankind, such as increased crop yields, quality or pathogen-resistance (Mason and Batley 2015). Cell volume generally rises with increasing genome size
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