Reduced, Posterior swim bladder inflation

The teleost swim bladder is a gas-filled structure that consists of two chambers, the posterior and anterior chamber. In zebrafish, the posterior chamber inflates around 96-120 h post fertilization (hpf) which is 2-3 days post hatch, and the anterior chamber inflates around 21 dpf (days post fertilization). In fathead minnow, the posterior and anterior chamber inflate around 6 and 14 dpf respectively.

The posterior chamber is formed from a bud originating from the foregut endoderm (Winata et al., 2009). The posterior chamber operates as a hydrostatic organ. The volume of gas in the adult swim bladder is continuously adjusted to regulate body density and buoyancy.

Many amphibians and frogs go through an embryo-larval transition phase marking the switch from endogenous feeding (from the yolk) to exogenous feeding. In zebrafish, embryonic-to-larval transition takes place around 96 hours post fertilization (hpf). As in amphibians, the transition between the different developmental phases includes maturation and inflation of the swim bladder (Liu and Chan, 2002).

Reduced inflation of the posterior chamber may manifest itself as either a complete failure to inflate the chamber or a reduced size of the chamber.

In several fish species, inflation of the posterior chamber can easily be observed using a stereomicroscope because the larvae are still transparent during those early developmental stages. This is for example true for zebrafish and fathead minnow. Posterior chamber size can then be measured based on photographs with a calibrator.

When observing effects on swim bladder inflation, it is important to verify that reduced swim bladder inflation occurs at concentrations significantly lower than those causing mortality, since a wide variety of chemicals cause impaired posterior chamber inflation at exposure concentrations that also cause mortality (Stinckens et al., 2018).

Taxonomic: Teleost fish can be divided in two groups according to swim bladder morphology: physoclistous (e.g., yellow perch, sea bass, striped bass, medaka) and physostomous (e.g., zebrafish and fathead minnow). Physostomous fish retain a duct between the digestive tract and the swim bladder during adulthood allowing them to gulp air at the surface to fill the swim bladder. In contrast, in physoclistous fish, once initial inflation by gulping atmospheric air at the water surface has occurred, the swim bladder is closed off from the digestive tract and swim bladder volume is regulated by gas secretion into the swim bladder (Wooley and Qin, 2010).

Much of the evidence for impaired posterior chamber of the swim bladder currently comes from work on zebrafish and fathead minnow (Stinckens et al., 2018; Cavallin et al., 2017; Wang et al., 2020). Increasing evidence is becoming available on defects of swim bladder inflation in medaka (Oryzias latipes), a species with only one swim bladder chamber (Gonzalez-Doncel et al., 2003; Dong et al., 2016; Kupsco et al., 2016; Mu et al., 2017; Pandelides et al., 2021). Exposure to T3, methimazole, heptafluorobutanoic acid (PFBA) and tris[1,3-dichloro-2-propyl] phosphate (TDCPP) inhibited inflation of the swim bladder in female medaka. Interestingly, for those females that developed a swim bladder, exposure to methimazole and all halogenated chemicals with the exception of PFBA, resulted in larger swim bladders (Godfrey et al., 2019). Horie et al. (2022) elucidated the timing of swim bladder inflation in medaka and compared effects on the swim bladder after exposure of zebrafish and medaka to PFBA and TDCPP. This KE is plausibly applicable across fish species with swim bladders, both physostomous and physoclistous.

Life stage: The posterior chamber inflates during a specific developmental time frame. In zebrafish, the posterior chamber inflates around 96-120 hpf which is 2-3 dph. In the fathead minnow, the posterior chamber inflates around 6 dpf. In medaka, the swim bladder inflates around 2 hours post hatch (hatching occurs around 8 dpf) (Horie et al., 2022). Therefore this KE is only applicable to the embryonic life stage.

Sex: This KE is plausibly applicable to both sexes. Sex differences are not often investigated in tests using early life stages of fish. In medaka, sex can be morphologically distinguished as soon as 10 days post fertilization. Females appear more susceptible to thyroid‐induced swim bladder dysfunction compared with males (Godfrey et al., 2019). In zebrafish and fathead minnow, it is currently unclear whether sex-related differences are important in determining the magnitude of the changes in this KE. Zebrafish are undifferentiated gonochorists since both sexes initially develop an immature ovary (Maack and Segner, 2003). Immature ovary development progresses until approximately the onset of the third week. Later, in female fish immature ovaries continue to develop further, while male fish undergo transformation of ovaries into testes. Final transformation into testes varies among male individuals, however finishes usually around 6 weeks post fertilization. Since the posterior chamber inflates around 5 days post fertilization in zebrafish, when sex differentiation has not started yet, sex differences are expected to play a minor role. Fathead minnow gonad differentiation also occurs during larval development. Fathead minnows utilize a XY sex determination strategy and markers can be used to genotype sex in life stages where the sex is not yet clearly defined morphologically (Olmstead et al., 2011). Ovarian differentiation starts at 10 dph followed by rapid development (Van Aerle et al., 2004). At 25 dph germ cells of all stages up to the primary oocytes stage were present and at 120 dph, vitellogenic oocytes were present. The germ cells (spermatogonia) of the developing testes only entered meiosis around 90–120 dph. Mature testes with spermatozoa are present around 150 dph. Since the posterior chamber inflates around 6 days post fertilization (1 dph) in fathead minnows, sex differences are expected to play a minor role in the current KE.

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