Triploidy in zebrafish larvae: Effects on gene expression, cell size and cell number, growth, development and swimming performance

Abstract

There is renewed interest in the regulation and consequences of cell size adaptations in studies on understanding the ecophysiology of ectotherms. Here we test if induction of triploidy, which increases cell size in zebrafish (Danio rerio), makes for a good model system to study consequences of cell size. Ideally, diploid and triploid zebrafish should differ in cell size, but should otherwise be comparable in order to be suitable as a model. We induced triploidy by cold shock and compared diploid and triploid zebrafish larvae under standard rearing conditions for differences in genome size, cell size and cell number, development, growth and swimming performance and expression of housekeeping genes and hsp70.1. Triploid zebrafish have larger but fewer cells, and the increase in cell size matched the increase in genome size (+ 50%). Under standard conditions, patterns in gene expression, ontogenetic development and larval growth were near identical between triploids and diploids. However, under demanding conditions (i.e. the maximum swimming velocity during an escape response), triploid larvae performed poorer than their diploid counterparts, especially after repeated stimuli to induce swimming. This result is consistent with the idea that larger cells have less capacity to generate energy, which becomes manifest during repeated physical exertion resulting in increased fatigue. Triploidy induction in zebrafish appears a valid method to increase specifically cell size and this provides a model system to test for consequences of cell size adaptation for the energy budget and swimming performance of this ectothermic vertebrate.

Shrinking body sizes in response to warming: explanations for the temperature–size rule with special emphasis on the role of oxygen

“…Across species or degrees of cell ploidy, cell size appears to be linked to the size of the nucleus, which in turn is linked to genome size, although the causality and its direction are not completely resolved (Gregory, 2001; Cavalier‐Smith, 2005; Hessen et al ., 2013). Indeed, artificially inducing triploidy in zebrafish ( Danio rerio ) resulted in a 50% increase in cell size, resembling the 50% increase in genome size (Van de Pol, Flik, & Verberk, 2020). Studies have found that plastic thermal responses in body size were accompanied by dynamic adjustments in both cell size and nucleus size (by adjusting chromatin packaging) and thus there is scope for cell size also to generate or parallel the TSR during ontogeny (Hermaniuk et al ., 2016; Leinaas et al ., 2016).…”

Early, nonlethal ploidy and genome size quantification using confocal microscopy in zebrafish embryos

“…The utility of myofiber nuclear volume analysis for identifying triploids is clear, but this technique has produced some curious observations that are difficult to explain. Diploid embryos consistently have one tight peak of nuclear volumes and a second, smaller peak of nuclei that are approximately double the size (perhaps pre‐mitotic cells in the process of DNA replication as suggested in Van de Pol et al, 2020). While triploid embryos do produce a peak nuclear volume approximately 50% larger than that of diploids (as expected), the values are not normally distributed, extending into and below the modal volume of diploid embryos.…”

“…Mortality rates were similar between their heat‐treated triploids and our pressure‐treated triploids until 48 hpf, after which mortality rate improved greatly in pressure‐treated embryos (our study) but not in heat‐treated embryos (Delomas & Dabrowski, 2018). In contrast, survival of cold‐treated zebrafish embryos (71%; presumptive triploids) was not significantly different from controls (77%; presumptive diploids) at 24 hpf (Van de Pol et al, 2020) confirming that mortality is increased in the first 24 h regardless of the technique used to manipulate ploidy. We have shown that if a pressure‐treated triploid embryo survives beyond 24 hpf, they are likely to be viable, but we are unable to discern between the true effects of increasing ploidy and the effects of the physical treatments on embryo survival from our study.…”

“…Triploid zebrafish have been produced by inhibiting extrusion of the second polar body via temperature treatment shortly after fertilization (Baars et al, 2016; Kavumpurath & Pandian, 1990; Van de Pol et al, 2020). Given that hydrostatic pressure is widely used to produce triploids of other species of fish (Piferrer et al, 2009) and has been used in zebrafish to produce gynogenetic homozygous diploids after fertilization with irradiated sperm (Streisinger et al, 1981), we reasoned that this technique would produce triploid zebrafish if we fertilized eggs with intact sperm.…”

“…Current techniques to determine ploidy level in zebrafish are lethal until fish are adult‐sized (approximately 3 months old) and, therefore, large enough that blood can be sampled for erythrocyte size measurement (Kavumpurath & Pandian, 1990). While flow cytometric measurement of cellular DNA content in cell suspensions made from embryos is a useful strategy to ensure that triploidy induction is successful at the population level (Delomas & Dabrowski, 2018; Van de Pol et al, 2020), this also requires destructive sampling and introduces the potential confound of competition among individuals of different ploidy within a tank (O'Keefe & Benfey, 1997; Taylor et al, 2014). Furthermore, many of the powerful molecular techniques available in zebrafish such as qPCR and RNAseq require embryo homogenization, making it challenging to validate ploidy in the embryos.…”