The ecological function of thyroid hormones.

Thyroid hormones (TH) are central hormonal regulators, orchestrating gene expression and complex biological processes vital for growth and reproduction in variable environments by triggering specific developmental processes in response to external cues. TH serve distinct roles in different species: inducing metamorphosis in amphibians or teleost fishes, governing metabolic processes in mammals, and acting as effectors of seasonality. These multifaceted roles raise questions about the underlying mechanisms of TH action. Recent evidence suggests a shared ecological role of TH across vertebrates, potentially extending to a significant portion of bilaterian species. According to this model, TH ensure that ontogenetic transitions align with environmental conditions, particularly in terms of energy expenditure, helping animals to match their ontogenetic transition with available resources. This alignment spans post-embryonic developmental transitions common to all vertebrates and more subtle adjustments during seasonal changes. The underlying logic of TH function is to synchronize transitions with the environment. This review briefly outlines the fundamental mechanisms of thyroid signalling and shows various ways in which animals use this hormonal system in natural environments. Lastly, we propose a model linking TH signalling, environmental conditions, ontogenetic trajectory and metabolism. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

The multi-level regulation of clownfish metamorphosis by thyroid hormones.

Most marine organisms have a biphasic life cycle during which pelagic larvae transform into radically different juveniles. In vertebrates, the role of thyroid hormones (THs) in triggering this transition is well known, but how the morphological and physiological changes are integrated in a coherent way with the ecological transition remains poorly explored. To gain insight into this question, we performed an integrated analysis of metamorphosis of a marine teleost, the false clownfish (Amphiprion ocellaris). We show how THs coordinate a change in color vision as well as a major metabolic shift in energy production, highlighting how it orchestrates this transformation. By manipulating the activity of liver X regulator (LXR), a major regulator of metabolism, we also identify a tight link between metabolic changes and metamorphosis progression. Strikingly, we observed that these regulations are at play in the wild, explaining how hormones coordinate energy needs with available resources during the life cycle.

Direct development of the catfish pectoral fin: An alternative pectoral fin pattern of teleosts.

BACKGROUND: The study of the teleosts' pectoral fin development touches on many crucial issues of evolutionary biology, from the formation of local adaptations to the tetrapod limbs' origin. Teleosts' pectoral fin is considered a rather developmentally and anatomically conservative structure. It displays larval and adult stages differing in the skeletal and soft tissues' composition. Larva-adult transition proceeds under the thyroid hormone (TH) control that defines pectoral fin ontogeny as an indirect development. However, the outstanding diversity of teleosts allows suggesting the existence of lineage specific developmental patterns. RESULTS: We present a description of the North African catfish, Clarias gariepinus, pectoral fin development. It lacks a clear larval stage and directly develops the adult skeleton with the associated musculature and innervation. Interestingly, the development of catfish pectoral fin appears not to be under the TH dependence. CONCLUSION: This catfish displays a direct pectoral fin developmental trajectory differing from the stereotyped teleost pattern. In the absence of the larval endoskeletal disk and TH control, the catfish's proximal radials arise in a manner somewhat similar to the metapterygial radials in basal actinopterygians and humerus in sarcopterygians. Thus, the catfish fin pattern seems homoplastic, arising by convergence with, or reversion to the ancestral developmental mechanisms.

Thyroid hormones regulate the formation and environmental plasticity of white bars in clownfishes.

Determining how plasticity of developmental traits responds to environmental conditions is a challenge that must combine evolutionary sciences, ecology, and developmental biology. During metamorphosis, fish alter their morphology and color pattern according to environmental cues. We observed that juvenile clownfish (Amphiprion percula) modulate the developmental timing of their adult white bar formation during metamorphosis depending on the sea anemone species in which they are recruited. We observed an earlier formation of white bars when clownfish developed with Stichodactyla gigantea (Sg) than with Heteractis magnifica (Hm). As these bars, composed of iridophores, form during metamorphosis, we hypothesized that timing of their development may be thyroid hormone (TH) dependent. We treated clownfish larvae with TH and found that white bars developed earlier than in control fish. We further observed higher TH levels, associated with rapid white bar formation, in juveniles recruited in Sg than in Hm, explaining the faster white bar formation. Transcriptomic analysis of Sg recruits revealed higher expression of duox, a dual oxidase implicated in TH production as compared to Hm recruits. Finally, we showed that duox is an essential regulator of iridophore pattern timing in zebrafish. Taken together, our results suggest that TH controls the timing of adult color pattern formation and that shifts in duox expression and TH levels are associated with ecological differences resulting in divergent ontogenetic trajectories in color pattern development.

The real Nemo movie: Description of embryonic development in Amphiprion ocellaris from first division to hatching.

BACKGROUND: Amphiprion ocellaris is one of the rare reef fish species that can be reared in aquaria. It is increasingly used as a model species for Eco-Evo-Devo. Therefore, it is important to have an embryonic development table based on high quality images that will allow for standardized sampling by the scientific community. RESULTS: Here we provide high-resolution time-lapse videos to accompany a detailed description of embryonic development in A ocellaris. We describe a series of developmental stages and we define six broad periods of embryogenesis: zygote, cleavage, blastula, gastrula, segmentation, and organogenesis that we further subdivide into 32 stages. These periods highlight the changing spectrum of major developmental processes that occur during embryonic development. CONCLUSIONS: We provide an easy system for the determination of embryonic stages, enabling the development of A ocellaris as a coral reef fish model species. This work will facilitate evolutionary development studies, in particular studies of the relationship between climate change and developmental trajectories in the context of coral reefs. Thanks to its lifestyle, complex behavior, and ecology, A ocellaris will undoubtedly become a very attractive model in a wide range of biological fields.

A star is born again: Methods for larval rearing of an emerging model organism, the False clownfish Amphiprion ocellaris.

As interest increases in ecological, evolutionary, and developmental biology (Eco-Evo-Devo), wild species are increasingly used as experimental models. However, we are still lacking a suitable model for marine fish species, as well as coral reef fishes that can be reared at laboratory scales. Extensive knowledge of the life cycle of anemonefishes, and the peculiarities of their biology, make them relevant marine fish models for developmental biology, ecology, and evolutionary sciences. Here, we present standard methods to maintain breeding pairs of the anemonefish Amphiprion ocellaris in captivity, obtain regular good quality spawning, and protocols to ensure larval survival throughout rearing. We provide a detailed description of the anemonefish husbandry system and life prey culturing protocols. Finally, a "low-volume" rearing protocol useful for the pharmacological treatment of larvae is presented. Such methods are important as strict requirements for large volumes in rearing tanks often inhibit continuous treatments with expensive or rare compounds.

Anemonefish, a model for Eco-Evo-Devo.

Anemonefish, are a group of about 30 species of damselfish (Pomacentridae) that have long aroused the interest of coral reef fish ecologists. Combining a series of original biological traits and practical features in their breeding that are described in this paper, anemonefish are now emerging as an experimental system of interest for developmental biology, ecology and evolutionary sciences. They are small sized and relatively easy to breed in specific husbandries, unlike the large-sized marine fish used for aquaculture. Because they live in highly structured social groups in sea anemones, anemonefish allow addressing a series of relevant scientific questions such as the social control of growth and sex change, the mechanisms controlling symbiosis, the establishment and variation of complex color patterns, and the regulation of aging. Combined with the use of behavioral experiments, that can be performed in the lab or directly in the wild, as well as functional genetics and genomics, anemonefish provide an attractive experimental system for Eco-Evo-Devo.

Anthropogenic stressors impact fish sensory development and survival via thyroid disruption.

Larval metamorphosis and recruitment represent critical life-history transitions for most teleost fishes. While the detrimental effects of anthropogenic stressors on the behavior and survival of recruiting fishes are well-documented, the physiological mechanisms that underpin these patterns remain unclear. Here, we use pharmacological treatments to highlight the role that thyroid hormones (TH) play in sensory development and determining anti-predator responses in metamorphosing convict surgeonfish, Acanthurus triostegus. We then show that high doses of a physical stressor (increased temperature of +3 °C) and a chemical stressor (the pesticide chlorpyrifos at 30 µg L-1) induced similar defects by decreasing fish TH levels and affecting their sensory development. Stressor-exposed fish experienced higher predation; however, their ability to avoid predation improved when they received supplemental TH. Our results highlight that two different anthropogenic stressors can affect critical developmental and ecological transitions via the same physiological pathway. This finding provides a unifying mechanism to explain past results and underlines the profound threat anthropogenic stressors pose to fish communities.

Sea anemone and clownfish microbiota diversity and variation during the initial steps of symbiosis.

Clownfishes and sea anemones form an intriguing long-term association, but the mechanism underlying this symbiosis is not well understood. Since clownfishes seem to cover themselves with sea anemone mucus, we investigated the microbiomes of the two partners to search for possible shifts in their compositions. We used a 16S rRNA gene sequencing strategy to study the dynamics of the microbiota during the association between the clownfish Amphiprion ocellaris and its host Heteractis magnifica under laboratory conditions. The experiment conducted in aquaria revealed that both clownfish and sea anemone mucus had specific signatures compared to artificial sea water. The microbiomes of both species were highly dynamic during the initiation of the symbiosis and for up to seven days after contact. Three families of bacteria (Haliangiaceae, Pseudoalteromonadacae, Saprospiracae) were shared between the two organisms after symbiosis. Once the symbiosis had been formed, the clownfishes and sea anemone then shared some communities of their mucus microbiota. This study paves the way for further investigations to determine if similar microbial signatures exist in natural environments, whether such microbial sharing can be beneficial for both organisms, and whether the microbiota is implicated in the mechanisms that protect the clownfish from sea anemone stinging.

[Metamorphosis of marine fish larvae and thyroid hormones]. / Les larves de poissons coralliens : un nouveau modèle d'étude de la métamorphose et des hormones thyroïdiennes.

Life history transitions are critical for many animal species and often correspond to concomitant developmental and ecological shifts. However, to date, little is known on how internal and external cues act together during these events. The life cycle of most teleostean reef fish includes a major developmental and ecological transition. Adults reproduce in the vicinity of the reef, emitting eggs that disperse and hatch in the ocean, where the larvae grow. Thereafter, larvae migrate back towards reefs where they settle and persist, at a step called larval recruitment. Larval recruitment involves the perception of environmental cues for larvae to localize and select their new benthic habitat, and is accompanied by major morphological changes. This ecological and developmental transition of pelagic larvae into reef-associated juveniles, often referred to as metamorphosis, are under the control of Thyroid Hormones (TH: T4, T3) and their receptors (TRαa, TRαb and TRß). This step is critical for the maintenance of reef fish populations, but its molecular control remains largely unknown. Recent results have brought new insights on coral reef fish metamorphosis. We have shown that TH and TR coordinate the metamorphosis that occurs during the entry in the reef of two coral reef fishes, the surgeon fish Acanthurus triostegus, and the clown fish Amphiprion ocellaris. We demonstrated an increase of TH-levels and TR-expression in the larvae, followed by a decrease in deriving juvenile. We observed similar trends (although with different dynamics and/or magnitude) in other coral reef fish species, therefore allowing us to generalize these observations. Interestingly, functional experiments such as treatments with pharmacological compounds exhibiting antagonist activity interfere with the surgeonfish and the clown fish larval transformation demonstrating a direct role of these hormones in controlling metamorphosis. All these results and in particular the dependency on thyroid hormones of the larval to juvenile transformation suggest that this step can be sensitive to disruption by environmental pollutants, such as endocrine disruptors. Using as model compound, chlorpyrifos, a pesticide often encountered in coral reefs, we showed that it impairs surgeonfish as well as clown fish transformation, hence diminishing the quality of the juvenile emerging from this transition. Larval recruitment in coral reef fish therefore corresponds to a TH-controlled metamorphosis, sensitive to endocrine disruption. Since metamorphosis and larval recruitment are essential for the maintenance of fish populations and subsequent coral reef resilience, it is important to better understand, at the molecular, anatomical and behavioral levels, how global changes and water pollution can threaten reef ecosystems.

Staging and normal table of postembryonic development of the clownfish (Amphiprion ocellaris).

BACKGROUND: The clownfish Amphiprion ocellaris is one of the rare coral reef fish species that can be reared in aquaria. With relatively short embryonic and larval development, it could be used as a model species to study the impact of global changes such as temperature rise or anthropogenic threats (eg, pollution) on the postembryonic development at molecular and endocrinological levels. Establishing a developmental table allows us to standardize sampling for the scientific community willing to conduct experiments on this species on different areas: ecology, evolution, and developmental biology. RESULTS: Here, we describe the postembryonic developmental stages for the clownfish A. ocellaris from hatching to juvenile stages (30 days posthatching). We quantitatively followed the postembryonic growth and described qualitative traits: head, paired and unpaired fins, notochord flexion, and pigmentation changes. The occurrence of these changes over time allowed us to define seven stages, for which we provide precise descriptions. CONCLUSIONS: Our work gives an easy system to determine A. ocellaris postembryonic stages allowing, thus, to develop this species as a model species for coral reef fishes. In light of global warming, the access to the full postembryonic development stages of coral reef fish is important to determine stressors that can affect such processes.

Insights into the Genomics of Clownfish Adaptive Radiation: Genetic Basis of the Mutualism with Sea Anemones.

Clownfishes are an iconic group of coral reef fishes, especially known for their mutualism with sea anemones. This mutualism is particularly interesting as it likely acted as the key innovation that triggered clownfish adaptive radiation. Indeed, after the acquisition of the mutualism, clownfishes diversified into multiple ecological niches linked with host and habitat use. However, despite the importance of this mutualism, the genetic mechanisms allowing clownfishes to interact with sea anemones are still unclear. Here, we used a comparative genomics and molecular evolutionary analyses to investigate the genetic basis of clownfish mutualism with sea anemones. We assembled and annotated the genome of nine clownfish species and one closely related outgroup. Orthologous genes inferred between these species and additional publicly available teleost genomes resulted in almost 16,000 genes that were tested for positively selected substitutions potentially involved in the adaptation of clownfishes to live in sea anemones. We identified 17 genes with a signal of positive selection at the origin of clownfish radiation. Two of them (Versican core protein and Protein O-GlcNAse) show particularly interesting functions associated with N-acetylated sugars, which are known to be involved in sea anemone discharge of toxins. This study provides the first insights into the genetic mechanisms of clownfish mutualism with sea anemones. Indeed, we identified the first candidate genes likely to be associated with clownfish protection form sea anemones, and thus the evolution of their mutualism. Additionally, the genomic resources acquired represent a valuable resource for further investigation of the genomic basis of clownfish adaptive radiation.

Developmental and comparative transcriptomic identification of iridophore contribution to white barring in clownfish.

Actinopterygian fishes harbor at least eight distinct pigment cell types, leading to a fascinating diversity of colors. Among this diversity, the cellular origin of the white color appears to be linked to several pigment cell types such as iridophores or leucophores. We used the clownfish Amphiprion ocellaris, which has a color pattern consisting of white bars over a darker body, to characterize the pigment cells that underlie the white hue. We observe by electron microscopy that cells in white bars are similar to iridophores. In addition, the transcriptomic signature of clownfish white bars exhibits similarities with that of zebrafish iridophores. We further show by pharmacological treatments that these cells are necessary for the white color. Among the top differentially expressed genes in white skin, we identified several genes (fhl2a, fhl2b, saiyan, gpnmb, and apoD1a) and show that three of them are expressed in iridophores. Finally, we show by CRISPR/Cas9 mutagenesis that these genes are critical for iridophore development in zebrafish. Our analyses provide clues to the genomic underpinning of color diversity and allow identification of new iridophore genes in fish.

Ontogenetic and phylogenetic simplification during white stripe evolution in clownfishes.

BACKGROUND: Biologists have long been fascinated by the striking diversity of complex color patterns in tropical reef fishes. However, the origins and evolution of this diversity are still poorly understood. Disentangling the evolution of simple color patterns offers the opportunity to dissect both ultimate and proximate causes underlying color diversity. RESULTS: Here, we study clownfishes, a tribe of 30 species within the Pomacentridae that displays a relatively simple color pattern made of zero to three vertical white stripes on a dark body background. Mapping the number of white stripes on the evolutionary tree of clownfishes reveals that their color pattern diversification results from successive caudal to rostral losses of stripes. Moreover, we demonstrate that stripes always appear with a rostral to caudal stereotyped sequence during larval to juvenile transition. Drug treatments (TAE 684) during this period leads to a dose-dependent loss of stripes, demonstrating that white stripes are made of iridophores and that these cells initiate the stripe formation. Surprisingly, juveniles of several species (e.g., Amphiprion frenatus) have supplementary stripes when compared to their respective adults. These stripes disappear caudo-rostrally during the juvenile phase leading to the definitive color pattern. Remarkably, the reduction of stripe number over ontogeny matches the sequences of stripe losses during evolution, showing that color pattern diversification among clownfish lineages results from changes in developmental processes. Finally, we reveal that the diversity of striped patterns plays a key role for species recognition. CONCLUSIONS: Overall, our findings illustrate how developmental, ecological, and social processes have shaped the diversification of color patterns during the radiation of an emblematic coral reef fish lineage.

Boat noise prevents soundscape-based habitat selection by coral planulae.

Understanding the relationship between coral reef condition and recruitment potential is vital for the development of effective management strategies that maintain coral cover and biodiversity. Coral larvae (planulae) have been shown to use certain sensory cues to orient towards settlement habitats (e.g. the odour of live crustose coralline algae - CCA). However, the influence of auditory cues on coral recruitment, and any effect of anthropogenic noise on this process, remain largely unknown. Here, we determined the effect of protected reef (MPA), exploited reef (non-MPA) soundscapes, and a source of anthropogenic noise (boat) on the habitat preference for live CCA over dead CCA in the planula of two common Indo-Pacific coral species (Pocillopora damicornis and Acropora cytherea). Soundscapes from protected reefs significantly increased the phonotaxis of planulae of both species towards live CCA, especially when compared to boat noise. Boat noise playback prevented this preferential selection of live CCA as a settlement substrate. These results suggest that sources of anthropogenic noise such as motor boat can disrupt the settlement behaviours of coral planulae. Acoustic cues should be accounted for when developing management strategies aimed at maximizing larval recruitment to coral reefs.

Decreased retention of olfactory predator recognition in juvenile surgeon fish exposed to pesticide.

Dory, the animated surgeonfish created by the Pixar Animation studios, famously suffered from short-term memory loss leading to many adventures. In reality, many fishes have excellent cognitive abilities and are able to learn and retain important information such as the identity of predators. However, if and how cognition can be affected by anthropogenically altered oceanic conditions is poorly understood. Here, we examine the effect of a widely used pesticide, chlorpyrifos, on the retention of acquired predator recognition in post-larval stage of the surgeonfish Acanthurus triostegus. Through associative learning, post-larvae of A. triostegus were first observed to forage significantly less in the presence of conspecific alarm cues and alarm cues associated to a predator's odor. The retention of this anti-predator behavior was estimated to last between 2 and 5 days in the absence of pesticide. However, environmentally-relevant concentrations of chlorpyrifos (1 µg.L-1) induced the loss of this acquired predator recognition. This reduced ability to recognize learned predators is discussed as it may lead to more vulnerable fish communities in coastal areas subjected to organophosphate pesticide pollution.

Exposure to agricultural pesticide impairs visual lateralization in a larval coral reef fish.

Lateralization, i.e. the preferential use of one side of the body, may convey fitness benefits for organisms within rapidly-changing environments, by optimizing separate and parallel processing of different information between the two brain hemispheres. In coral reef-fishes, the movement of larvae from planktonic to reef environments (recruitment) represents a major life-history transition. This transition requires larvae to rapidly identify and respond to sensory cues to select a suitable habitat that facilitates survival and growth. This 'recruitment' is critical for population persistence and resilience. In aquarium experiments, larval Acanthurus triostegus preferentially used their right-eye to investigate a variety of visual stimuli. Despite this, when held in in situ cages with predators, those larvae that previously favored their left-eye exhibited higher survival. These results support the "brain's right-hemisphere" theory, which predicts that the right-eye (i.e. left-hemisphere) is used to categorize stimuli while the left-eye (i.e. right-hemisphere) is used to inspect novel items and initiate rapid behavioral-responses. While these experiments confirm that being highly lateralized is ecologically advantageous, exposure to chlorpyrifos, a pesticide often inadvertently added to coral-reef waters, impaired visual-lateralization. This suggests that chemical pollutants could impair the brain function of larval fishes during a critical life-history transition, potentially impacting recruitment success.

Evolution of ligands, receptors and metabolizing enzymes of thyroid signaling.

Thyroid hormones (THs) play important roles in vertebrates such as the control of the metabolism, development and seasonality. Given the pleiotropic effects of thyroid disorders (developmental delay, mood disorder, tachycardia, etc), THs signaling is highly investigated, specially using mammalian models. In addition, the critical role of TH in controlling frog metamorphosis has led to the use of Xenopus as another prominent model to study THs action. Nevertheless, animals regarded as non-model species can also improve our understanding of THs signaling. For instance, studies in amphioxus highlighted the role of Triac as a bona fide thyroid hormone receptor (TR) ligand. In this review, we discuss our current understanding of the THs signaling in the different taxa forming the metazoans (multicellular animals) group. We mainly focus on three actors of the THs signaling: the ligand, the receptor and the deiodinases, enzymes playing a critical role in THs metabolism. By doing so, we also pinpoint many key questions that remain unanswered. How can THs accelerate metamorphosis in tunicates and echinoderms while their TRs have not been yet demonstrated as functional THs receptors in these species? Do THs have a biological effect in insects and cnidarians even though they do not have any TR? What is the basic function of THs in invertebrate protostomia? These questions can appear disconnected from pharmacological issues and human applications, but the investigation of THs signaling at the metazoans scale can greatly improve our understanding of this major endocrinological pathway.

Chemical spying in coral reef fish larvae at recruitment.

When fish larvae recruit back to a reef, chemical cues are often used to find suitable habitat or to find juvenile or adult conspecifics. We tested if the chemical information used by larvae was intentionally produced by juvenile and adult conspecifics already on the reef (communication process) or whether the cues used result from normal biochemical processes with no active involvement by conspecifics ("spying" behavior by larvae). Conspecific chemical cues attracted the majority of larvae (four out of the seven species tested); although while some species were equally attracted to cues from adults and juveniles (Chromis viridis, Apogon novemfasciatus), two exhibited greater sensitivity to adult cues (Pomacentrus pavo, Dascyllus aruanus). Our results indicate also that spying cues are those most commonly used by settling fishes (C. viridis, P. pavo, A. novemfasciatus). Only one species (D. aruanus) preferred the odour of conspecifics that had had visual contact with larvae (communication).