Relationship: 1026: Inhibition, Deiodinase 2 leads to Decreased, Triiodothyronine (T3) in serum

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic: Deiodinases are important for the activation of T4 to T3 across vertebrates. There appear to be differences among vertebrate classes relative to the role of the different deiodinase isoforms in regulating thyroid hormone levels. Maia et al. (2005) determined that in a normal physiological situation in humans the contribution of DIO2 to plasma T3 levels is twice that of DIO1. A DIO2 knockout (KO) mouse however showed a very mild gross phenotype with only mild growth retardation in males (Schneider et al., 2001). It seemed that by blocking the negative feedback system, DIO2 KO resulted in increased levels of T4 and TSH and in normal rather than decreased T3 levels compared to WT. Potential differences in the role of the deiodinase isoforms in the negative feedback system and the final consequences for TH levels across vertebrates is currently not entirely clear. These differences make it difficult to exactly evaluate the importance of DIO2 in regulating serum/tissue T3 levels across vertebrates. Mol et al. (1998) concluded that deiodinases in teleosts were more similar to mammalian deiodinases than had been generally accepted, based on the similarities in susceptibility to inhibition and the agreement of the Km values.

Life stage and sex: Deiodinases are important for the activation of T4 to T3 across all life stages and sexes.

Key Event Relationship Description

The two major thyroid hormones are thyroxine (T4) and the more biologically active triiodothyronine (T3), both iodinated derivatives of tyrosine. Active and inactive THs are tightly regulated by enzymes called iodothyronine deiodinases (DIO). The activation occurs via outer ring deiodination (ORD), i.e. removing iodine from the outer, phenolic ring of T4 to form T3, while inactivation occurs via inner ring deiodination (IRD), i.e. removing iodine from the inner tyrosol ring of T4 or T3.

Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of TH action. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain selenocysteine in their catalytic centre. Type I deiodinase is capable of converting T4 into T3, as well as to convert rT3 to the inactive thyroid hormone 3,3’ T2, through outer ring deiodination. rT3, rather than T4, is the preferred substrate for DIO1. furthermore, DIO1 has a very high Km (µM range, compared to nM range for DIO2) (Darras and Van Herck, 2012). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate (i.e., activation of T4 tot T3). DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, reverse T3, (rT3) and 3,3’-T2 respectively. (Darras and Van Herck, 2012)

Evidence Supporting this KER

Inhibition of DIO2 activity is widely accepted to directly decrease T3 levels, since the conversion of T4 to T3 is inhibited. The importance of DIO2 inhibition in altering serum T3 levels depends on the relative role of different deiodinases in regulating serum versus tissue T3 levels and in negative feedback within the HPT axis. Both aspects appear to vary among vertebrate taxa.

Inhibition of DIO2 activity is widely accepted to directly decrease T3 levels, since the conversion of T4 to T3 is inhibited.

• Houbrechts et al. (2016) developed a Dio2 knockout and confirmed both the absence of the full length Dio2 protein in the liver and the dramatical decrease of T4 activating enzyme activity in liver, brain and eyes. Finally, they found decreased levels of T3 in liver, brain and eyes.
• Winata et al. (2009, 2010) reported reduced pigmentation, otic vesicle length and head-trunk angle in DIO1+2 and DIO2 knockdown fish. These effects were rescued after T3 supplementation but not by T4 supplementation, confirming that decreased T3 levels were at the basis of the observed effects.
• In the study of Cavallin et al. (2017) fathead minnow larvae were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). Transcriptional analysis showed that especially DIO2, but also DIO3 mRNA levels (in some treatments), were increased in 10 to 21 day old larvae exposed to IOP as of the age of 6 days. This suggests that IOP effectively inhibited DIO2 and DIO3 in the larvae and that mRNA levels increased as a compensatory response. The authors also observed pronounced decreases of whole body T3 concentrations and increases of whole body T4 concentrations.
• Stinckens et al. (2020) showed that IOP reduced T3 levels in zebrafish in 21 and 32 day old larvae that had been exposed starting from fertilization.
• While DIO1 has a high Km and rT3 is its preferred substrate, DIO2 has a low Km and T4 is its preferred substrate, indicating that DIO2 is more important than DIO1 in converting T4 to T3 in a physiological situation (Darras and Van Herck, 2012).

Since in fish early life stages THs are typically measured on a whole body level, it is currently uncertain whether T3 level changes occur at the serum and/or tissue level. Pending more dedicated studies, whole body TH levels are considered a proxy for serum TH levels. 

The importance of DIO2 inhibition in altering serum T3 levels depends on the relative role of different deiodinases in regulating serum versus tissue T3 levels and in negative feedback within the HPT axis. Both aspects appear to vary among vertebrate taxa. The high level of DIO2 activity and its expression in the liver of teleosts are unique among vertebrates (Orozco and Valverde, 2005). It is thought that DIO2 is important for local T3 production in several tissues but also contributes to circulating T3, especially in fish and amphibians (Darras et al., 2015).

In DIO2 knockout mice it seemed that the negative feedback system was blocked resulting in increased levels of T4 and TSH and in normal rather than decreased T3 levels compared to WT.

In the study of Cavallin et al. (2017) fathead minnow embryos were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). The authors observed increased whole body T3 concentrations in 4 and 6 day old embryos, while they observed decreased T3 concentrations in 10 to 21 day old larvae exposed to IOP as of the age of 6 days. One possible explanation for the elevated T3 concentrations may be the potential impact of IOP exposure on DIO3. DIO3 is an inactivating enzyme that removes iodine from the inner ring of both T4 and T3, resulting in reverse T3 (rT3) and 3,5-diiodo-L-thyronine (T2), respectively (Bianco and Kim, 2006). Maternal sources of thyroid hormones are known to include both T4 and T3 (Power et al., 2001; Walpita et al., 2007). Consequently, reduced conversion of maternal T3 to inactive forms may be one plausible explanation for the increase. Another explanation may result from the role of deiodinases in the negative feedback system of the HPT axis. Inibition of deiodinase (unclear which isoforms) may block the negative feedback system and result in increased release of T4. Increased levels of T4 were indeed observed by Cavallin et al. (2017).

Quantitative Understanding of the Linkage

Since in fish enzyme activity and thyroid hormone levels are rarely measured in the same study, quantitative understanding of this linkage is limited.

Thyroid hormone levels are regulated via negative feedback, influencing this KER. Additionally, deiodinases regulate the activity of thryoid hormones, not only in serum and target organs, but also in the thryoid gland. Deiodinases themselves are known to be involved in the negative feedback system that results in increased TSH levels when the levels of T4 (and also T3) in serum are low (Schneider et al., 2001), resulting in an even more complicated impact on this KER. Increased TSH levels then stimulate increased T4 release from the thyroid gland, resulting in a compensatory increase of serum T4 levels. In DIO2 knockout mice it seemed that the negative feedback system was blocked resulting in increased levels of T4 and TSH and in normal rather than decreased T3 levels compared to WT. By inhibiting DIO1 using a PTU exposure, Schneider et al. (2001) showed that DIO2 played a role in the increased TSH levels in response to T3 or T4 injection.

References

Bianco, A.C., Kim, B.W., 2006. Deiodinases: implications of the local control of thyroid hormone action. Journal of Clinical Investigation 116, 2571-2579.

Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.

Darras, V.M., Van Herck, S.L.J., 2012. Iodothyronine deiodinase structure and function: from ascidians to humans. Journal of Endocrinology 215, 189-206.

Darras, V.M., Houbrechts, A.M., Van Herck, S.L.J., 2015. Intracellular thyroid hormone metabolism as a local regulator of nuclear thyroid hormone receptor-mediated impact on vertebrate development.

Houbrechts, A.M., Delarue, J., Gabriels, I.J., Sourbron, J., Darras, V.M., 2016. Permanent Deiodinase Type 2 Deficiency Strongly Perturbs Zebrafish Development, Growth, and Fertility. Endocrinology 157, 3668-3681.

Maia, A.L., Kim, B.W., Huang, S.A., Harney, J.W., Larsen, P.R., 2005. Type 2 iodothyronine deiodinase is the major source of plasma T-3 in euthyroid humans. Journal of Clinical Investigation 115, 2524-2533.

Mol, K.A., Van der Geyten, S., Burel, C., Kuhn, E.R., Boujard, T., Darras, V.M., 1998. Comparative study of iodothyronine outer ring and inner ring deiodinase activities in five teleostean fishes. Fish Physiology and Biochemistry 18, 253-266.

Orozco, A., Valverde, R.C., 2005. Thyroid hormone deiodination in fish. Thyroid 15, 799-813.

Power, D.M., Llewellyn, L., Faustino, M., Nowell, M.A., Bjornsson, B.T., Einarsdottir, I.E., Canario, A.V., Sweeney, G.E., 2001. Thyroid hormones in growth and development of fish. Comp Biochem Physiol C Toxicol Pharmacol 130, 447-459.

Schneider, M.J., Fiering, S.N., Pallud, S.E., Parlow, A.F., St Germain, D.L., Galton, V.A., 2001. Targeted disruption of the type 2 selenodeiodinase gene (D102) results in a phenotype of pituitary resistance to T-4. Molecular Endocrinology 15, 2137-2148.

Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.

Walpita, C.N., Van der Geyten, S., Rurangwa, E., Darras, V.M., 2007. The effect of 3,5,3'-triiodothyronine supplementation on zebrafish (Danio rerio) embryonic development and expression of iodothyronine deiodinases and thyroid hormone receptors. Gen Comp Endocrinol 152, 206-214.

Winata, C.L., Korzh, S., Kondrychyn, I., Korzh, V., Gong, Z. 2010. The role of vasulature and blood circulation in zebrafish swim bladder development. Dev. Biol. 10:3.

Winata, C.L., Korzh, S., Kondrychyn, I., Zheng, W., Korzh, V., Gong, Z. 2009. Development of zebrafish swimbladder: the requirement of Hedgehog signaling in specification and organization of the three tissue layers. Dev. Biol.331, 222–236, http://dx.doi.org/10.1016/j.ydbio.2009.04.035.

Relationship: 1027: Decreased, Triiodothyronine (T3) in serum leads to Reduced, Posterior swim bladder inflation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic: The indirect relationship between deiodinase inhibition, expected to lead to reduced T3 levels, and reduced posterior chamber inflation has been confirmed in early zebrafish larvae around the time of posterior chamber inflation, i.e. around 5 days post fertilization in zebrafish (Stinckens et al., 2018) and around 6 days post fertilization in fathead minnows (Cavallin et al., 2017).

Life stage: This KER is only applicable to early embryonic development, which is the period where the posterior swim bladder chamber inflates. The relationship between reduced T3 levels and reduced posterior chamber inflation is not applicable to older larvae that successfully inflated the posterior chamber but show impaired anterior chamber inflation after chronic exposure to low concentrations of thyroid hormone system disruptors. In 32 day old zebrafish exposed to methimazole, propylthiouracil, 2-mercaptobenzothiazole or iopaonic acid (Stinckens et al., 2016, 2020) as well as in 14-21 day old fathead minnows exposed to iopaonic acid (Cavallin et al., 2017), a clear inverse relationship was found. With decreasing whole body T3 concentrations, posterior chamber volume increased, suggesting a possible compensatory mechanism for the observed decrease in anterior chamber volume. As a result, the sum of both chamber surfaces, reflecting the total amount of gas, was equal to controls for most treatments (Stinckens et al., 2016; Stinckens et al., 2020).

Sex: 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, when sex differentiation has not started yet, sex differences are expected to play a minor role.

Key Event Relationship Description

Reduced T3 levels in serum prohibit local TH action in the target tissues. Since swim bladder development and/or inflation is regulated by thyroid hormones, this results in impaired posterior chamber inflation.

Evidence Supporting this KER

There is convincing evidence that decreased T3 levels result in impaired posterior chamber inflation, but the underlying mechanisms are not completely understood. The quantitative understanding is currently very limited because T3 levels and posterior inflation are seldom measured in the same study. Therefore the evidence supporting this KER can be considered moderate.

Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition (Liu and Chan, 2002) and larval-to-juvenile transition (Brown et al., 1997) in fish. Inflation of the posterior chamber is part of the embryonic-to-larval transition in fish, together with structural and functional maturation of the mouth and gastrointestinal tract, and resorption of the yolk sac (Liu and Chan, 2002). Marelli et al. (2016) showed that thyroid hormone receptor alpha and beta are both expressed in swim bladder tissue of zebrafish at 5 days post fertilization, corresponding to the timing of posterior inflation. this time point has additionally been shown to coincide with increased T3 and T4 levels (Chang et al., 2012), suggesting that posterior inflation is under thyroid hormone regulation.

• Maternal injection of T3, resulting in increased T3 concentrations in the eggs of striped bass (Morone saxatilis) lead to significant increases in both swim bladder inflation and survival (Brown et al., 1988).
• Dong et al. (2013) and Thisse et al. (2003) showed localized expression of DIO1 and DIO2 in the swim bladder tissue of 96 and 120 hpf zebrafish larvae, suggesting that local activation of thryoid hormones (i.e. conversion of T4 to T3) is requried in swim bladder tissue around that time period.
• Marelli et al. (2016) used morpholinos to block translation of thryoid hormone receptor alpha or beta in zebrafish. They found that thyroid hormone receptor alpha and beta knockdowns failed to inflate the posterior chamber of the swim bladder by 120 hpf, indicating that the action of T3 is needed for proper inflation of the posterior chamber. High T3 doses partially rescued the negative impact in partially resistant mutants, further confirming the importance of T3 in this process.
• Stinckens et al. (2018) showed that effects on posterior chamber inflation in zebrafish could be predicted based on in chemico DIO2 inhibition potential with only few false positives and false negatives. While T3 levels were not determined in this study, DIO2 inhibition is expected to result in decreased T3 levels.
• Bagci et al. (2015) and Heijlen et al. (2013, 2014) reported that knockdown of DIO1+2 in zebrafish resulted in impairment of the inflation of the posterior chamber of the swim bladder. DIO1 and 2 knockdown is expected to result in reduced T3 levels. Indeed, Walpita et al. (2009, 2010) showed that T3 supplementation effectively rescued the effects of DIO1 and 2 knockdown, while T4 supplementation did not.
• de Vrieze et al. (2014) found that knockdown of monocarboxylate transporter 8 (mct8) in zebrafish resulted in a dose-dependent impairment of posterior chamber inflation. Since this transporter is known to transport thyroid hormones across cell membranes, this supports the importance of thyroid hormones in regulating posterior chamber inflation.
• Shi et al. (2019) found that exposure of adult zebrafish to 6:2 chlorinated polyfluorinated ether sulfonate (F-53B), an alternative to perfluorooctanesulfonate (PFOS), decreased T3 levels in both male and female zebrafish. Additionally,  F-53B was maternally transferred to the offspring. Decreased T3 levels together with impaired posterior chamber inflation was observed in the F1 offspring.
• Wang et al. (2020) observed a decrease of whole-body T3 as well as impaired posterior chamber inflation in zebrafish exposed to perfluorooctanoic acid and perfluoropolyether carboxylic acids from fertilization until the age of 5 days. Exogeneous T3 or T4 supplementation partly rescued PFECA-induced posterior swim bladder malformation, confirming the causal relationship between reduced T3 levels and reduced posterior chamber inflation.
• Molla et al. (2019) showed that T3 supplementation increased posterior chamber diameter in zebrafish larvae. This confirms that T3 plays an important role in posterior swim bladder inflation.

The mechanism through which altered TH levels result in impaired posterior chamber inflation still needs to be elucidated. It is currently unclear which aspect of swim bladder development and inflation is affected by TH disruption. Based on the developmental stages of the posterior chamber, several hypotheses could explain effects on posterior chamber inflation due to disrupted TH levels. A first hypothesis includes effects on the budding of the posterior chamber inflation. Secondly, the effect on posterior chamber inflation could also be caused by disturbing the formation and growth of the three tissue layers of this organ. It has been reported that the Hedgehog signalling pathway plays an essential role in swim bladder development and is required for growth and differentiation of cells of the swim bladder. The Wnt/β-catenin signalling pathway is required for the organization and growth of all three tissue layers (Yin et al., 2011, 2012, Winata 2009, Kress et al., 2009). Both signalling pathways have been related to THs in amphibian and rodent species (Kress et al., 2009; Plateroti et al., 2006; Stolow and Shi, 1995). Molla et al. (2019) showed that insulin-like growth factor (IGF‐1) plays a role in swim bladder inflation/maturation in zebrafish. Several other hypotheses include effects on the successful initial inflation of the posterior chamber, effects on lactic acid production that is required for the maintenance of the swim bladder volume, or effects on the production of surfactant that is crucial to maintain the surface tension necessary for swim bladder inflation.

Another uncertainty lies in the relative importance of the different T4 actvating iodothyronine deiodinases (DIO1, DIO2) in regulating swim bladder inflation. Stinckens et al. (2018) showed that exposure of zebrafish embryos to seven strong DIO1 inhibitors (measured using in chemico enzyme inhibition assays), six out of seven compounds impaired posterior chamber inflation. Exposure to strong DIO2 inhibitors on the other hand affected posterior chamber inflation and/or surface area in all cases. These results suggest that DIO2 enzymes may play a more important role in swim bladder inflation compared to DIO1 enzymes. it has been previously suggested that DIO2 is the major contributor to TH activation in developing zebrafish embryos (Darras et al., 2015; Walpita et al., 2010). It has been shown that a morpholino knockdown targeting DIO1 mRNA alone did not affect embryonic development in zebrafish, while knockdown of DIO2 delayed progression of otic vesicle length, head-trunk angle and pigmentation index (Houbrechts et al., 2016; Walpita et al., 2010, 2009). DIO1 inhibition may only become essential in hypothyroidal circumstances, for example when DIO2 is inhibited or in case of iodine deficiency, in zebrafish (Walpita et al., 2010) and mice (Galton et al., 2009; Schneider et al., 2006).

As reported by Bagci et al. (2015) and Heijlen et al. (2014), posterior chamber inflation was impaired in DIO3 knockdown zebrafish. Heijlen et al. (2014) additionally reported histologically abnormal tissue layers in the swim bladder of DIO3 knockdown zebrafish. DIO3 is a thyroid hormone inactivating enzyme, which would result in higher levels of T3 in serum. Wei et al. (2018) showed that exposure to bisphenol S in adult zebrafish decreased T4 levels and increased T3 levels, and these changes in thyroid hormone levels were transferred to the offspring, in which impaired swim bladder inflation was observed. This indicates that not only too low, but also too high T3 levels, impact posterior chamber inflation. The underlying mechanism is currently unknown.

In the study of Cavallin et al. (2017) fathead minnow embryos were exposed to IOP, a model iodothyronine deiodinase inhibitor that is assumed to inhibit all three deiodinase enzymes (DIO1,2,3). The authors observed increased whole body T3 concentrations in 4 and 6 day old embryos, together with impaired posterior chamber inflation. Transcript levels of DIO1, 2 and 3 remained unaltered and thus offered no proof of a compensatory mechanism that could explain these results.

The earliest life stages of teleost fish rely on maternally transferred THs to regulate certain developmental processes until embryonic TH synthesis is active (Power et al., 2001). As a result, posterior swim bladder chamber inflation, which occurs early during development, appears to be less sensitive to inhibition of TH synthesis than to inhibition of the conversion of T4 to T3 (Stinckens et al., 2016, 2018; Nelson et al., 2016). There have however been a few reports of reduced posterior inflation upon inhibition of TH synthesis (Liu and Chan, 2002). It must however be noted that these observations could reflect delayed inflation due to a general delay in development rather than a direct effect on the swim bladder. Longer observations would have to clarify this.

References

Bagci, E., Heijlen, M., Vergauwen, L., Hagenaars, A., Houbrechts, A.M., Esguerra, C.V., Blust, R., Darras, V.M., Knapen, D., 2015. Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction. PLOS One 10, e0123285.

Brown, C.L., Doroshov, S.I., Nunez, J.M., Hadley, C., Vaneenennaam, J., Nishioka, R.S., Bern, H.A., 1988. MATERNAL TRIIODOTHYRONINE INJECTIONS CAUSE INCREASES IN SWIMBLADDER INFLATION AND SURVIVAL RATES IN LARVAL STRIPED BASS, MORONE-SAXATILIS. Journal of Experimental Zoology 248, 168-176.

Brown, D.D., 1997. The role of thyroid hormone in zebrafish and axolotl development. Proceedings of the National Academy of Sciences of the United States of America 94, 13011-13016.

Cavallin, J.E., Ankley, G.T., Blackwell, B.R., Blanksma, C.A., Fay, K.A., Jensen, K.M., Kahl, M.D., Knapen, D., Kosian, P.A., Poole, S.T., Randolph, E.C., Schroeder, A.L., Vergauwen, L., Villeneuve, D.L., 2017. Impaired swim bladder inflation in early life stage fathead minnows exposed to a deiodinase inhibitor, iopanoic acid. Environmental Toxicology and Chemistry 36, 2942-2952.

Chang, J., Wang, M., Gui, W., Zhao, Y., Yu, L., Zhu, G., 2012. Changes in Thyroid Hormone Levels during Zebrafish Development. Zoological Science 29, 181-184.

Darras, V.M., Houbrechts, A.M., Van Herck, S.L.J., 2015. Intracellular thyroid hormone metabolism as a local regulator of nuclear thyroid hormone receptor-mediated impact on vertebrate development. Biochimica Et Biophysica Acta-Gene Regulatory Mechanisms 1849, 130-141.

de Vrieze, E., van de Wiel, S.M.W., Zethof, J., Flik, G., Klaren, P.H.M., Arjona, F.J., 2014. Knockdown of Monocarboxylate Transporter 8 (mct8) Disturbs Brain Development and Locomotion in Zebrafish. Endocrinology 155, 2320-2330.

Dong, W., Macaulay, L.J., Kwok, K.W., Hinton, D.E., Stapleton, H.M., 2013. Using whole mount in situ hybridization to examine thyroid hormone deiodinase expression in embryonic and larval zebrafish: a tool for examining OH-BDE toxicity to early life stages. Aquat Toxicol 132-133, 190-199.

Galton, V.A., Schneider, M.J., Clark, A.S., St Germain, D.L., 2009. Life without thyroxine to 3,5,3'-triiodothyronine conversion: studies in mice devoid of the 5'-deiodinases. Endocrinology 150, 2957-2963.

Heijlen, M., Houbrechts, A., Bagci, E., Van Herck, S., Kersseboom, S., Esguerra, C., Blust, R., Visser, T., Knapen, D., Darras, V., 2014. Knockdown of type 3 iodothyronine deiodinase severely perturbs both embryonic and  early larval development in zebrafish. Endocrinology 155, 1547-1559.

Heijlen, M., Houbrechts, A.M., Darras, V.M., 2013. Zebrafish as a model to study peripheral thyroid hormone metabolism in vertebrate development. Gen.Comp. Endocrinol. 188, 289–296, http://dx.doi.org/10.1016/j.ygcen.2013.04.004.

Kress, E., Rezza, A., Nadjar, J., Samarut, J., Plateroti, M., 2009. The frizzled-related sFRP2 gene is a target of thyroid hormone receptor alpha1 and activates beta-catenin signaling in mouse intestine. J Biol Chem 284, 1234-1241.

Liu, Y.W., Chan, W.K., 2002. Thyroid hormones are important for embryonic to larval transitory phase in zebrafish. Differentiation 70, 36-45.

Maack, G., Segner, H., 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology 62, 895-906.

Marelli, F., Carra, S., Agostini, M., Cotelli, F., Peeters, R., Chatterjee, K., Persani, L., 2016. Patterns of thyroid hormone receptor expression in zebrafish and generation of a novel model of resistance to thyroid hormone action. Molecular and Cellular Endocrinology 424, 102-117.

Molla, M.H.R., Hasan, M.T., Jang, W.J., Diaz, C.D.S., Appenteng, P., Marufchoni, H., Jahan, B., Brown, C.L., 2019. Thyroid hormone-induced swim bladder and eye maturation are transduced by IGF-1 in zebrafish embryos. Aquaculture Research 50, 3462-3470.

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Plateroti, M., Kress, E., Mori, J.I., Samarut, J., 2006. Thyroid hormone receptor alpha1 directly controls transcription of the beta-catenin gene in intestinal epithelial cells. Mol Cell Biol 26, 3204-3214.

Schneider, M.J., Fiering, S.N., Thai, B., Wu, S.Y., St Germain, E., Parlow, A.F., St Germain, D.L., Galton, V.A., 2006. Targeted disruption of the type 1 selenodeiodinase gene (Dio1) results in marked changes in thyroid hormone economy in mice. Endocrinology 147, 580-589.

Shi, G., Wang, J., Guoa, H., Shenga, N., Cui, Q., Pan, Y., Guob, Y., Sun, Y., Dai, J., 2019. Parental exposure to 6:2 chlorinated polyfluorinated ether sulfonate (F-53B) induced transgenerational thyroid hormone disruption in zebrafish. Science of the Total Environment 665, 855-863.

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Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.

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Walpita, C.N., Crawford, A.D., Janssens, E.D., Van der Geyten, S., Darras, V.M., 2009. Type 2 iodothyronine deiodinase is essential for thyroid hormone-dependent embryonic development and pigmentation in zebrafish. Endocrinology 150, 530-539.

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Wei, P.H., Zhao, F., Zhang, X.N., Liu, W.M., Jiang, G.B., Wang, H.F., Ru, S.G., 2018. Transgenerational thyroid endocrine disruption induced by bisphenol S affects the early development of zebrafish offspring. Environmental Pollution 243, 800-808.

Winata, C.L., Korzh, S., Kondrychyn, I., Zheng, W., Korzh, V., Gong, Z., 2009. Development of zebrafish swimbladder: The requirement of Hedgehog signaling in specification and organization of the three tissue layers. Developmental Biology 331, 222-236.

Yin, A., Korzh, S., Winata, C.L., Korzh, V., Gong, Z., 2011. Wnt signaling is required for early development of zebrafish swimbladder. PLoS One 6, e18431.

Yin, A., Korzh, V., Gong, Z., 2012. Perturbation of zebrafish swimbladder development by enhancing Wnt signaling in Wif1 morphants. Biochim Biophys Acta 1823, 236-244.

Relationship: 1028: Reduced, Posterior swim bladder inflation leads to Reduced, Swimming performance

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic: Importance of proper functioning of the swim bladder for supporting natural swimming behaviour can be plausibly assumed to be generally applicable to fish possessing a posterior chamber. Evidence exists for a wide variety of freshwater and marine fish species.

Life stage: This KER is only applicable to early embryonic development, which is the period where the posterior swim bladder chamber inflates. To what extent fish can survive and swim with partly inflated swim bladders during later life stages is unknown.

Sex: 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, when sex differentiation has not started yet, sex differences are expected to play a minor role.

Key Event Relationship Description

Effects on swim bladder inflation can alter swimming performance and buoyancy of fish, which is essential for predator avoidance, energy sparing, migration, reproduction and feeding behaviour, resulting in lower young-of-year survival.

Evidence Supporting this KER

The weight of evidence supporting a direct linkage between these two KEs, i.e. reduced posterior swim bladder inflation and reduced swimming performance, is moderate.

The posterior chamber of the swim bladder has a function in regulating the buoyancy of fish (Roberston et al., 2007). Fish rely on the lipid and gas content in their body to regulate their position within the water column, with the latter being more efficient at increasing body buoyancy. Therefore, fish with functional swim bladders have no problem supporting their body (Brix 2002), while it is highly likely that impaired inflation severely impacts swimming performance, as has been suggested previously (Bagci et al., 2015; Hagenaars et al., 2014). Fish without a functional swim bladder are severely disadvantaged, making the likelihood of surviving smaller. Stoyek et al. (2011) showed that the posterior chamber volume is maintained at a stable level at varying pressures corresponding to varying depths through gas exchange with the anteror chamber.

Buoyancy is one of the primary mechanisms of fish to regulate behaviour, swimming performance and energy expenditure. There is extensive evidence of a link between reduced posterior chamber inflation and reduced swimming performance:

• Stewart and Gee (1981) showed that fathead minnows swimming from still water to a current resorbed gas to fill the swim bladder and tailor buoyancy precisely to the level were swimming is most efficient.
• Lindsey et al., 2010 reported that zebrafish larvae that fail to inflate their swim bladder use additional energy to maintain buoyancy (Lindsey et al., 2010, Goodsell et al., 1996), possibly contributing to reduced swimming activity. Furthermore, they reported that the range of swimming depth varies with stages of swim bladder development.
• Czesny et al., 2005 reported that yellow perch larvae without inflated swim bladders capture free-swimming prey poorly and expend more energy on feeding and maintaining their position within the water column, due to impacted swimming behaviour.
• Kurata et al., 2014 observed that Bluefin tuna larvae present at the bottom of a tank, incapable of swimming upwards, had significantly lower swim bladder inflation.
• Chatain (1994) associated sea bass larvae with non-inflated swim bladders with numerous complications, such as spinal deformities and lordosis and reduced growth rates, adding to the impact on swimming behaviour.
• An increasing incidence of swim bladder non-inflation has also been reported in Atlantic salmon. Affected fish had severely altered balance and buoyancy, observed through a specific swimming behaviour, as the affected fish were swimming upside down in an almost vertical position (Poppe et al., 1997).
• Permanent DIO 2 deficiency in zebrafish was shown to result in reduced posterior chamber inflation and disturbed locomotor activity (Houbrechts et al., 2016).
• Michiels et al. (2017) showed that both for controls and zebrafish embryos exposed to an environmental sample, the swimming distance was significantly lower in larvae that failed to inflate the posterior chamber compared to larvae from the same treatment that had inflated posterior chambers.
• Exposure of zebrafish embryos to thyroid disrupting compounds resulted in an effect on posterior chamber inflation as well as on the swimming distance in the larval stage (Stinckens et al., unpublished).
• All zebrafish larvae that failed to inflate the posterior chamber after exposure to 2 mg/L iopanoic acid (IOP), died by the age of 9 dpf (Stinckens et al., 2020). Since larvae from the same group that were able to inflate the posterior chamber survived, it is plausible to assume that uninflated posterior chambers limited the ability to swim and find food.
• Hagenaars et al. (2014) showed that zebrafish embryos exposed to 4.28 mg/L PFOS had lower swimming speeds when the posterior chamber was not inflated. It should be noted that almost all larvae with a non-inflated swimbladder had a spinal curvature and it could therefore not statistically be determined whether the reduced swimming speed was due to a spinal curvature, a non-inflated swim bladder or the interaction of both.
• Knockdown of deiodinase 3 (expected to lead to hyperthyroidism) in zebrafish was shown to result in both impaired inflation of the posterior chamber and reduced swimming activity and escape response (Heijlen et al., 2014; Bagci et al., 2015).
• Massei et al. (in preparation) showed that impaired swim bladder inflation and reduced swimming activity of 5 day old zebrafish larvae were correlated after exposure to narcotics.

Robertson et al., (2007) reported that the swim bladder only becomes functional as a buoyancy regulator when it is fully developed into a double-chambered swim bladder. This implies that effects on posterior chamber inflation would not directly result in effects on swimming capacity. However, it was also reported that gas in the swim bladder increases the buoyancy of zebrafish larvae already just after initial inflation, while it would be actively controlled only after 28–30 d post hatch. Therefore, an effect on swimming capacity is still likely.

Exposure of zebrafish embryos to 6-propylthiouracil (PTU) resulted in an effect on posterior chamber inflation, but did not result in a direct effect on the swimming distance in the larval stage (Stinckens et al., unpublished). Vergauwen et al. (2015) reported decreased swimming activity as well as impaired posterior chamber inflation after exposure to phenanthrene, a non-polar narcotic, but there was no significant difference between swimming activity of larvae with our without inflated posterior chamber within the same treatment. Possibly, the impact of baseline toxicity on respiration and energy metabolism was more important in decreasing swimming activity compared to impaired inflation of the posterior chamber.

It has been difficult to unambiguously attribute reduced swimming activity to impaired inflation of the posterior chamber, since swimming activity can be altered via different modes of action including altered energy metabolism, altered brain development and thus swimming behaviour. For example, the swimming activity of zebrafish larvae was reduced after 5 days of exposure to 2-mercaptobenzothiazole (MBT), while they had inflated posterior chambers.

Quantitative Understanding of the Linkage

The quantitative understanding of the linkage between impaired posterior chamber inflation and effect on swimming behaviour is limited.

Relations between reduced swim bladder inflation and reduced swimming performance are currently based on a binary observation of swim bladder inflation. Several studies have shown that larvae with inflated swim bladders have higher swiming activity compared to larvae that failed to inflate the swim bladder. No direct relationship between swim bladder surface (quantitative measure of swim bladder inflation) and swimming performance has been reported yet.

The data of Michiels et al. (2017) and Stinckens et al. (unpublished) on swim bladder inflation and swimming activity have been collected on the same day. The process of posterior chamber inflation normally occurs during a specific developmental time frame, resulting in limited flexibility to explore temporal concordance. Based on the biologically plausible direct importance of swim bladder functionality to swimming performance, no lag is expected.

References

Bagci, E., Heijlen, M., Vergauwen, L., Hagenaars, A., Houbrechts, A.M., Esguerra, C.V.,Blust, R., Darras, V.M., Knapen, D., 2015. Deiodinase knockdown during earlyzebrafish development affects growth, development, energy metabolism,motility and phototransduction. PLoS One 10, e0123285, http://dx.doi.org/10.1371/journal.pone.0123285.

Brix O (2002) The physiology of living in water. In: Hart PJ, Reynolds J (eds) Handbook of Fish Biology and Fisheries, Vol. 1, pp. 70–96. Blackwell Publishing, Malden, USA.

Chatain, B., 1994. Abnormal swimbladder development and lordosis in sea bass (Dicentrarchus labrax) and sea bream (Sparus auratus). Aquaculture 119:371–379.

Czesny, S.J., Graeb, B.D.S., Dettmersn, J.M., 2005. Ecological consequences of swimbladder noninflation for larval yellow perch. Trans. Am. Fish. Soc. 134,1011–1020, http://dx.doi.org/10.1577/T04-016.1.

Goodsell, D.S., Morris, G.M., Olsen, A.J. 1996. Automated docking of fleixble ligands. Applications of Autodock. J. Mol. Recogonition, 9:1-5.

Hagenaars, A., Stinckens, E., Vergauwen, L., Bervoets, L., Knapen, D., 2014. PFOS affects posterior swim bladder chamber inflation and swimming performanceof zebrafish larvae. Aquat. Toxicol. 157, 225–235, http://dx.doi.org/10.1016/j.aquatox.2014.10.017.

Heijlen, M., Houbrechts, A., Bagci, E., Van Herck, S., Kersseboom, S., Esguerra, C., Blust, R., Visser, T., Knapen, D., Darras, V., 2014. Knockdown of type 3 iodothyronine deiodinase severely perturbs both embryonic and  early larval development in zebrafish. Endocrinology 155, 1547-1559.

Houbrechts, A.M., Delarue, J., Gabriels, I.J., Sourbron, J., Darras, V.M., 2016. Permanent Deiodinase Type 2 Deficiency Strongly Perturbs Zebrafish Development, Growth, and Fertility. Endocrinology 157, 3668-3681.

Kurata, M., Ishibashi, Y., Takii, K., Kumai, H., Miyashita, S., Sawada, Y., 2014.Influence of initial swimbladder inflation failure on survival of Pacific bluefintuna, Thuunus orientalis (Temminck and Schlegl) larvae. Aquacult. Res. 45,882–892.

Lindsey, B.W., Smith, F.M., Croll, R.P., 2010. From inflation to flotation: contributionof the swimbladder to whole-body density and swimming depth duringdevelopment of the zebrafish (Danio rerio). Zebrafish 7, 85–96, http://dx.doi.org/10.1089/zeb.2009.0616.

Maack, G., Segner, H., 2003. Morphological development of the gonads in zebrafish. Journal of Fish Biology 62, 895-906.

Massei R et al. (in preparation) Sublethal adverse effects of non-polar narcotics in the zebrafish embryo.

Michiels, E.D.G., Vergauwen, L., Hagenaars, A., Fransen, E., Van Dongen, S., Van Cruchten, S.J., Bervoets, L., Knapen, D., 2017. Evaluating Complex Mixtures in the Zebrafish Embryo by Reconstituting Field Water Samples: A Metal Pollution Case Study. International Journal of Molecular Sciences 18, 539.

Poppe, T.T., Hellberg, H., Griffiths, D., Mendal, H. 1977. Swim bladder abnormality in farmed Atlantic salmon, Salmo salar. Diseases of aquatic organisms 30:73-76.

Roberston, G.N., McGee, C.A.S., Dumbarton, T.C., Croll, R.P., Smith, F.M., 2007.Development of the swim bladder and its innervation in the zebrafish, Danio rerio. J. Morphol. 268, 967–985, http://dx.doi.org/10.1002/jmor.

Stewart, D.B., Gee, J.H., 1981. Mechanisms of buoyancy adjustments and effects of water velocity and temperature on ability to maintain buoyancy in fathead minnows, Pimephales promelas, Rafinesque. Comparative Biochemistry and Physiology a-Physiology 68, 337-347.

Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.

Stinckens, E., Vergauwen, L., Schroeder, A.L., Maho, W., Blackwell, B., Witter, H.,Blust, R., Ankley, G.T., Covaci, A., Villenueve, D.L., Knapen, D., 2016. Disruption of thyroid hormone balance after 2-mercaptobenzothiazole exposure causes swim bladder inflation impairment—part II: zebrafish. Aquat. Toxicol. 173:204-17.

Stoyek, M.R., Smith, F.M., Croll, R.P., 2011. Effects of altered ambient pressure on the volume and distribution of gas within the swimbladder of the adult zebrafish, Danio rerio. Journal of Experimental Biology 214, 2962-2972.

Vergauwen, L., Schmidt, S.N., Stinckens, E., Maho, W., Blust, R., Mayer, P., Covaci, A., Knapen, D., 2015. A high throughput passive dosing format for the Fish Embryo Acute Toxicity test. Chemosphere 139, 9-17.

Relationship: 2212: Reduced, Swimming performance leads to Increased Mortality

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Importance of swimming performance on survival is generally applicable to all hatched fish across life stages and sexes.

Key Event Relationship Description

Reduced swimming performance is likely to affect essential endpoints such as predator avoidance, feeding behaviour and reproduction. These parameters are biologicaly plausible to affect survival, especially in a non-laboratory environment where food is scarce and predators are abundant.

Evidence Supporting this KER

A direct relationship between reduced swimming performance and reduced survival is difficult to establish. There is however a lot of indirect evidence linking reduced swim bladder inflation to reduced survival (https://aopwiki.org/relationships/2213), which can be plausibly assumed to be related to reduced swimming performance.

For example, all zebrafish larvae that failed to inflate the posterior chamber after exposure to 2 mg/L iopanoic acid (IOP), died by the age of 9 dpf (Stinckens et al., 2020). Since larvae from the same group that were able to inflate the posterior chamber survived and the test was performed in the laboratory in optimal conditions, it is plausible to assume that the cause of death was the inability to swim and find food due to the failure to inflate the posterior swim bladder chamber.

Reduced swimming performance is likely to affect essential endpoints such as predator avoidance, feeding behaviour and reproduction. These parameters are biologicaly plausible to affect survival, especially in a non-laboratory environment where food is scarce and predators are abundant.

A direct relationship between reduced swimming performance and reduced survival is difficult to establish. There is however a lot of indirect evidence linking reduced swim bladder inflation to reduced survival (see non-adjacent KER 1041), which can be plausibly assumed to be related to reduced swimming performance.

For example, all zebrafish larvae that failed to inflate the posterior chamber after exposure to 2 mg/L iopanoic acid (IOP), died by the age of 9 dpf (Stinckens et al., 2020). Since larvae from the same group that were able to inflate the posterior chamber survived and the test was performed in the laboratory in optimal conditions, it is plausible to assume that the cause of death was the inability to swim and find food due to the failure to inflate the posterior swim bladder chamber.

A direct relationship between reduced swimming performance and reduced survival is difficult to establish in a laboratory environment where food is abundant and there are no predators.

Quantitative Understanding of the Linkage

Quantitative understanding of this linkage is currently limited.

Reduced swimming performance is not expected to immediately lead to mortality. Depending on the extent of the reduction in swimming performance and depending on the cause of death (e.g., starvation due to the inability to find food, being caught by a predator) the lag time may vary.

As an example, Stinckens et al. (2020) found that zebrafish larvae that failed to inflate the swim bladder at 5 dpf and did not manage to inflate it during the days afterwards died by the age of 9 dpf. Since zebrafish initiate exogenous feeding around day 5 when the yolk is almost completely depleted, there was a lag period of around 4 days after which reduced feeding resulted in mortality. Obviously, in a laboratory setup there is no increased risk of being caught by a predator.

References

Stinckens, E., Vergauwen, L., Blackwell, B.R., Anldey, G.T., Villeneuve, D.L., Knapen, D., 2020. Effect of Thyroperoxidase and Deiodinase Inhibition on Anterior Swim Bladder Inflation in the Zebrafish. Environmental Science & Technology 54, 6213-6223.

Relationship: 2013: Increased Mortality leads to Decrease, Population trajectory

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic: All organisms must survive to reproductive age in order to reproduce and sustain populations. The additional considerations related to young-of-year survival made above are applicable to other fish species in addition to zebrafish and fathead minnows with the same reproductive strategy (r-strategist as described in the theory of MaxArthur and Wilson (1967)). The impact of reduced young of year survival on population size is even greater for k-strategists that invest more energy in a lower number of offspring.

Life stage: Density dependent effects start to play a role in the larval stage of fish when free-feeding starts (Hazlerigg et al., 2014).

Sex: This linkage is independent of sex.

Key Event Relationship Description

• Increased mortality in the reproductive population may lead to a declining population.
• Increased mortality in the young-of-year cohort may lead to a declining population. This depends on the excess mortality due to the applied stressor and the environmental parameters such as food availability and predation rate. Most fish species are r-strategist, meaning they produce a lot of offspring instead of investing in parental care. This results in natural high larval mortality causing only a small percentage of the larvae to survive to maturity. If the excess larval mortality due to a stressor is small, the population dynamics might result in constant population size. Should the larval excess be more significant, or last on the long-term, this will affect the population. To calculate the long-term persistence of the population, population dynamic models should be used.

Evidence Supporting this KER

Survival rate is an obvious determinant of population size and is therefore included in population modeling (e.g., Miller et al., 2020).

• Survival to reproductive maturity is a parameter of demographic significance. Assuming resource availability (i.e., food, habitat, etc.) is not limiting to the extant population, sufficient mortality in the reproductive population may ultimately lead to declining population trajectories.
• Under some conditions, reduced larval survival may be compensated by reduced predation and increased food availability, and therefore not result in population decline (Stige et al., 2019).

• According to empirical data, combined with population dynamic models, feeding larvae are the crucial life stage in zebrafish (and other r-strategists) for the regulation of the population. (Schäfers et al., 1993)
• In fathead minnow, natural survival of young-of-year has been found to be highly variable and influential on population growth (Miller and Ankley, 2004)

• The extent to which larval mortality affects population size could depend on the fraction of surplus mortality compared to a natural situation.

• There are scenarios in which individual mortality may not lead to declining population size. These include instances where populations are limited by the availability of habitat and food resources, which can be replenished through immigration. Effects of mortality in the larvae can be compensated by reduced competition for resources (Stige et al., 2019).

• The direct impact of pesticides on migration behavior can be difficult to track in the field, and documentation of mortality during migration is likely underestimated (Eng 2017).

• In general, there is not enough empirical data on the relationships between survival and population level effects in fish (Rearick et al., 2018) to optimize population models.

References

Beaudouin, R., Goussen, B., Piccini, B., Augustine, S., Devillers, J., Brion, F., Pery, A.R., 2015. An individual-based model of zebrafish population dynamics accounting for energy dynamics. PloS one 10, e0125841.

Caswell, H., 2000. Matrix population models. Sinauer Sunderland, MA, USA.

Eng, M.L., Stutchbury, B.J.M. & Morrissey, C.A. Imidacloprid and chlorpyrifos insecticides impair migratory ability in a seed-eating songbird. Sci Rep 7, 15176 (2017)

Hazlerigg, C.R., Lorenzen, K., Thorbek, P., Wheeler, J.R., Tyler, C.R., 2012. Density-dependent processes in the life history of fishes: evidence from laboratory populations of zebrafish Danio rerio. PLoS One 7, e37550.

MacArthur, R., Wilson, E., 1967. The Theory of Island Biogeography. Princeton: Princeton Univ. Press. 203 p.

Miller, D.H., Ankley, G.T., 2004. Modeling impacts on populations: fathead minnow (Pimephales promelas) exposure to the endocrine disruptor 17β-trenbolone as a case study. Ecotoxicology and Environmental Safety 59, 1-9.

Miller, D.H., Clark, B.W., Nacci, D.E. 2020. A multidimensional density dependent matrix population model for assessing risk of stressors to fish populations. Ecotoxicology and environmental safety 201, 110786

Pinceel, T., Vanschoenwinkel, B., Brendonck, L., Buschke, F., 2016. Modelling the sensitivity of life history traits to climate change in a temporary pool crustacean. Scientific reports 6, 29451.

Rearick, D.C., Ward, J., Venturelli, P., Schoenfuss, H., 2018. Environmental oestrogens cause predation-induced population decline in a freshwater fish. Royal Society open science 5, 181065.

Schäfers, C., Oertel, D., Nagel, R., 1993. Effects of 3, 4-dichloroaniline on fish populations with differing strategies of reproduction. Ecotoxicology and Ecophysiology, 133-146.

Stige, L.C., Rogers, L.A., Neuheimer, A.B., Hunsicker, M.E., Yaragina, N.A., Ottersen, G., Ciannelli, L., Langangen, Ø., Durant, J.M., 2019. Density‐and size‐dependent mortality in fish early life stages. Fish and Fisheries 20, 962-976.Hazlerigg, C.R.E., Tyler, C.R., Lorenzen, K., Wheeler, J.R., Thorbek, P., 2014. Population relevance of toxicant mediated changes in sex ratio in fish: An assessment using an individual-based zebrafish (Danio rerio) model. Ecological Modelling 280, 76-88.

Stige, L.C., Rogers, L.A., Neuheimer, A.B., Hunsicker, M.E., Yaragina, N.A., Ottersen, G., Ciannelli, L., Langangen, O., Durant, J.M., 2019. Density- and size-dependent mortality in fish early life stages. Fish and Fisheries 20, 962-976.

Relationship: 1042: Inhibition, Deiodinase 2 leads to Reduced, Posterior swim bladder inflation

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic: The evidence for a relationship between DIO2 inhibition and inflation of the posterior chamber of the swim bladder is currently based on work in zebrafish and fathead minnow but is expected to be broadly applicable to fish.

Sex: This KER is probably not sex-dependent since both females and males rely on activation of THs by deiodinase for regulation of vital processes. Additionally, zebrafish are undifferentiated gonochorists, and gonad differentiation starts only around 23-25 dpf (Uchida et al., 2002), well after the time point of posterior chamber inflation (around 5 dpf).

Life stage: This KER is only applicable to early embryonic development, which is the period where the posterior swim bladder chamber inflates.

Key Event Relationship Description

The two major thyroid hormones are thyroxine (T4) and the more biologically active triiodothyronine (T3), both iodinated derivatives of tyrosine. Active and inactive THs are tightly regulated by enzymes called iodothyronine deiodinases (DIO). The activation occurs via outer ring deiodination (ORD), i.e. removing iodine from the outer, phenolic ring of T4 to form T3, while inactivation occurs via inner ring deiodination (IRD), i.e. removing iodine from the inner tyrosol ring of T4 or T3.

Three types of iodothyronine deiodinases (DIO1-3) have been described in vertebrates that activate or inactivate THs and are therefore important mediators of TH action. All deiodinases are integral membrane proteins of the thioredoxin superfamily that contain selenocysteine in their catalytic centre. Type I deiodinase is capable of converting T4 into T3, as well as to convert rT3 to the inactive thyroid hormone 3,3’ T2, through outer ring deiodination. rT3, rather than T4, is the preferred substrate for DIO1. furthermore, DIO1 has a very high Km (µM range, compared to nM range for DIO2) (Darras and Van Herck, 2012). Type II deiodinase (DIO2) is only capable of ORD activity with T4 as a preferred substrate (i.e., activation of T4 tot T3). DIO3 can inner ring deiodinate T4 and T3 to the inactive forms of THs, reverse T3, (rT3) and 3,3’-T2 respectively. (Darras and Van Herck, 2012)

Inhibition of DIO2 therefore results in decreased T3 levels. Since swim bladder development and/or inflation is regulated by thyroid hormones, this results in impaired posterior chamber inflation.

Evidence Supporting this KER

There is convincing evidence that inhibition of DIO activity, either through specific knockdown or through chemical exposure, results in impaired posterior chamber inflation, but the underlying mechanisms are not completely understood, including the relative importance of DIO1 and DIO2. Based on current evidence, it seems that DIO2 is more important in regulating posterior chamber inflation. Due to the difficulty of measuring DIO activity in small fish embryos, quantitative linkages and temporal concordance have been difficult to establish. The quantitative understanding is currently based on a relationship between the classification of chemicals according to their in chemico DIO inhibitory potential (using a threshold and uncertainty zone) on the one hand, and occurence of in vivo effects on posterior chamber inflation on the other hand. Predictions based on this relationship have been proven highly successful. Therefore the evidence supporting this KER can be considered moderate.

Inhibition of DIO 2 activity is widely accepted to reduce the conversion of T4 to the more biologically active T3. Thyroid hormones are known to be involved in development, especially in metamorphosis in amphibians and in embryonic-to-larval transition and larval-to-juvenile transition in fish. Inflation of the posterior swim bladder chamber is part of the embryonic-to-larval transition in fish, together with structural and functional maturation of the mouth and gastrointestinal tract, and resorption of the yolk sac. Together with empirical evidence, it is plausible to assume that posterior swim bladder inflation is under thyroid hormone regulation but scientific understanding is incomplete. It follows that disrupted conversion of T4 to T3 is likely to interfere with normal inflation of the posterior swim bladder chamber.

Deiodinases are criticial for normal development. Several defects have already been reported in cases where the TH hormone balance is disturbed. Winata et al. (2009, 2010) reported reduced pigmentation, otic vesicle length and head-trunk angle in DIO1+2 and DIO2 knockdown fish. These effects were rescued after T3 supplementation, indicating the importance of T4 to T3 conversion by deiodinases.

Substantial evidence for the link between deiodinase inhibition and impaired posterior chamber inflation is available:

• Chang et al., (2012) established a base-line for TH levels during zebrafish development and observed peaks in whole-body T3 content at 5 dpf when the posterior chamber of the swim bladder inflates.
• Bagci et al. (2015) and Heijlen et al. (2013, 2014) reported that knockdown of DIO1+2 in zebrafish resulted in impairment of the inflation of the posterior chamber of the swim bladder.
• Permanent DIO 2 deficiency in zebrafish was shown to result in reduced posterior chamber inflation (Houbrechts et al., 2016).
• DIO1 and DIO2 mRNA has also been shown to be present in zebrafish swim bladder tissue at 96 hpf using whole mount in situ hybridization (Heijlen et al., 2013; Dong et al., 2013), suggesting a tissue-specific role of T3 in the inflation process of the posterior chamber.
• Exposure to PTU, a very potent DIO1 inhibitor, caused thyroid hypertrophy in X. leavis because of the inhibition of the peripheral conversion of T4 to T3 (Degitz et al., 2005). PTU also decreased serum T3 levels in the rat (Frumess and Larsen, 1975) and resulted in effects on posterior chamber inflation in zebrafish (Jomaa et al., 2014; Stinckens et al., 2018). It should be noted that there are some uncertainties related to the species-specific susceptibility of DIO1 to inhibition by PTU, as teleostean DIO1 seems to be less sensitive to inhibition by PTU (Orozco and Valverde, 2005; Kuiper et al., 2006; Orozco et al., 2012).
• Stinckens et al. (2018) showed that effects on posterior chamber inflation in zebrafish could be predicted based on in chemico DIO2 inhibition potential with only few false positives and false negatives.
• After exposure of fathead minnows (Pimephales promelas) to the non-specific deiodinase inhibitor IOP from 1-6 dpf, Incidence and length of inflated posterior swim bladders were significantly reduced (Cavallin et al., 2017).
• While DIO1 has a high Km and rT3 is its preferred substrate, DIO2 has a low Km and T4 is its preferred substrate, indicating that DIO2 is more important than DIO1 in converting T4 to T3 in a physiological situation (Darras and Van Herck, 2012). It follows that DIO2 inhibition is likely more important than DIO1 inhibition in reducing posterior chamber inflation.

The mechanism through which altered TH levels result in impaired posterior chamber inflation still needs to be elucidated.

It is currently unclear which aspect of swim bladder development and inflation is affected by TH disruption. Based on the developmental stages of the posterior chamber, several hypotheses could explain effects on posterior chamber inflation due to disrupted TH levels. A first hypothesis includes effects on the budding of the posterior chamber inflation. Secondly, the effect on posterior chamber inflation could also be caused by disturbing the formation and growth of the three tissue layers of this organ. It has been reported that the Hedgehog signalling pathway plays an essential role in swim bladder development and is required for growth and differentiation of cells of the swim bladder. The Wnt/β-catenin signalling pathway is required for the organization and growth of all three tissue layers (Yin et al., 2011, 2012, Winata 2009, Kress et al., 2009). Both signalling pathways have been related to THs in amphibian and rodent species (Kress et al., 2009; Plateroti et al., 2006; Stolow and Shi, 1995). Several other hypotheses include effects on the successful initial inflation of the posterior chamber, effects on lactic acid production that is required for the maintenance of the swim bladder volume, or effects on the production of surfactant that is crucial to maintain the surface tension necessary for swim bladder inflation.

Another uncertainty lies in the relative importance of the different T4 activating iodothyronine deiodinases (DIO1, DIO2) in regulating swim bladder inflation. Stinckens et al. (2018) showed that when exposing zebrafish embryos to seven strong DIO1 inhibitors (measured using in chemico enzyme inhibition assays), six out of seven compounds impaired posterior chamber inflation. Exposure to strong DIO2 inhibitors on the other hand affected posterior chamber inflation and/or surface area in all cases. These results suggest that DIO2 enzymes may play a more important role in swim bladder inflation compared to DIO1 enzymes. it has been previously suggested that DIO2 is the major contributor to TH activation in developing zebrafish embryos (Darras et al., 2015; Walpita et al., 2010). It has been shown that a morpholino knockdown targeting DIO1 mRNA alone did not affect embryonic development in zebrafish, while knockdown of DIO2 delayed progression of otic vesicle length, head-trunk angle and pigmentation index (Houbrechts et al., 2016; Walpita et al., 2010, 2009). DIO1 inhibition may only become essential in hypothyroidal circumstances, for example when DIO2 is inhibited or in case of iodine deficiency, in zebrafish (Walpita et al., 2010) and mice (Galton et al., 2009; Schneider et al., 2006).

Heijlen et al. (2015) reported histologically abnormal tissue layers in the swim bladder of DIO3 knockdown zebrafish. As reported in Bagci et al. (2015) and Heijlen et al. (2014), posterior chamber inflation was impaired in DIO3 knockdown zebrafish. DIO3 is a thyroid hormone inactivating enzyme, which would result in higher levels of T3 in serum. This indicates that not only too low, but also too high T3 levels, impact posterior chamber inflation. The underlying mechanism is currently unknown.

References

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Heijlen, M., Houbrechts, A.M., Darras, V.M., 2013. Zebrafish as a model to study peripheral thyroid hormone metabolism in vertebrate development. Gen.Comp. Endocrinol. 188, 289–296, http://dx.doi.org/10.1016/j.ygcen.2013.04.004.

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Relationship: 2213: Reduced, Posterior swim bladder inflation leads to Increased Mortality

AOPs Referencing Relationship

Evidence Supporting Applicability of this Relationship

Taxonomic: The literature provides strong support for the relevance of this KER for physoclistous fish (e.g., yellow perch, Japanese Medaka) whose inflation occurs at a critical time in development when the fish must gulp air to inflate its swim bladder before the pneumatic duct closes. The relevance to physostomes (such as zebrafish and fathead minnows) that maintain an open pneumatic duct into adulthood is less apparent. The latter likely have greater potential to inflate the swim bladder at some point in development, even if early larval inflation is impaired. However, it is plausible that structural damage that prevented inflation of the organ in a phystostome would be expected to cause similar effects.

Life stage: This KER is applicable to early embry-larval development, which is the period where the posterior swim bladder chamber inflates and larvae start to freely feed. To what extent fish can survive with partly inflated swim bladders during later life stages is unknown.

Sex: This KER is probably not sex-dependent since both females and males rely on the posterior swim bladder chamber to regulate buyoancy. Furthermore, 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, when sex differentiation has not started yet, sex differences are expected to play a minor role.

Key Event Relationship Description

Because of its roles in energy sparing and swimming performance, it is expected that failure to inflate the swim bladder would create increased oxygen and energy demands leading to decreased growth, which in turn leads to decreased probability of survival.

Evidence Supporting this KER

The posterior chamber of the swim bladder has a function in regulating the buoyancy of fish (Roberston et al., 2007). Fish rely on the lipid and gas content in their body to regulate their position within the water column. Efficient regulation of buoyancy is energy sparing and allows for fish to expend less energy in maintaining and changing positions in the water column. Because of its roles in energy sparing and swimming performance, it is expected that failure to inflate the swim bladder would create increased oxygen and energy demands leading to decreased growth, which in turn leads to decreased probability of survival. In particular, these impacts would be expected in non-laboratory environments where fish must expend energy to capture food and avoid predators and where available food is limited. Additionally, fish without a functional swim bladder are severely disadvantaged in terms of foraging and avoiding predators, making the likelihood of surviving smaller.

• Czesny et al. (2005) demonstrated that swim bladder non-inflation was associated with multiple phenotypic and behavioral outcomes that would be expected to adversely impact young of year survival.
  • Yellow perch with non-inflated swim bladders grew more slowly than those with inflated swim bladders, both in the laboratory and in the field.
  • Yellow perch with non-inflated swim bladders always captured prey less efficiently than those with inflated swim bladders of the same size class.
  • Yellow perch with non-inflated swim bladders suffered from increased predation risk.
  • Yellow perch with non-inflated swim bladders experienced significantly increased mortality and lower time to mortality in a foodless environment compared to those with inflated swim bladders, indicating greater energy expenditure.
  • Yellow perch with non-inflated swim bladders had significantly greater oxygen consumption than fish of the same size class with inflated swim bladders, again indicating greater energy expenditure.
  • The authors hypothesized that failed swim bladder inflation occurs frequently in natural systems, but these individuals rarely survive in a natural environment where food resources are limited.
  • Note: yellow perch are a physoclistous species in which initial inflation can only occur during a narrow window of development in which the pneumatic duct is still connected to the gut, allowing the fish to gulp air and inflate its swim bladder. Once the pneumatic duct closes, normal inflation is no longer possible.
• In aquaculture systems, failure to inflate the swim bladder has been shown to reduce growth rates and cause high mortalities in a wide range of species (reviewed by Woolley and Qin, 2010).
• Pond-cultured walleye with non-inflated swim bladders were found to be smaller (weight and length) than fish with inflated swim bladders. There was also association with deformities (e.g., lordosis) that were expected to impair survival (Kindschi and Barrows, 1993).
• Review of failed swim bladder inflation in wild perch and 26 other physoclistous species showed that fish whose swim bladders failed to inflate had higher mortality, reduced growth, and increased incidence of spinal malformations stereotypical of persistent upward swimming (Egloff, 1996).
• Chatain (1994) reported that sea bream (Sparus auratus) and sea bass (Dicentrarchus labrax) with non-inflated swim bladders were 20-30% less in weight than those with inflated swim bladders and more susceptible to stress-induced mortality (e.g., associated with handling, hypoxia, etc.). It was suggested this was due to both increased energetic demands and decreased feeding efficiency.
• Marty et al. 1995 measured increased oxygen consumption in Japanese medaka (Oryzias latipes) with non-inflated swim bladders compared to those whose swim bladders had inflated.
• In zebrafish (Danio rerio) whose smim bladder inflation was prevented by holding in a closed chamber (preventing air gulping to inflate the swim bladder), larval survival was significantly less than that of fish held in open chambers whose swim bladders could inflate. There was also increased incidence of spinal curvature in the closed chamber fish whose swim bladders were prevented from inflating (Goolish and Oukutake, 1999).
• Maternal injection of T3, resulting in increased T3 concentrations in the eggs of striped bass (Morone saxatilis) lead to significant increases in both swim bladder inflation and survival (Brown et al., 1988).
• In striped bass, (Morone saxatilis) failure to inflate the swimbladder was reported to results in dysfunctional buoyancy control, deformities, and poor larval survival and growth (Martin-Robichaud and Peterson, 2008).
• All zebrafish larvae that failed to inflate the posterior chamber after exposure to 2 mg/L iopanoic acid (IOP), died by the age of 9 dpf (Stinckens et al., 2020). Since larvae from the same group that were able to inflate the posterior chamber survived, it is plausible to assume that uninflated posterior chambers limited the ability to swim and find food.

References