The Crustacean Ecdysone Cassette: a Gatekeeper for Molt and Metamorphosis

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The crustacean ecdysone cassette: a gatekeeper for molt and metamorphosis

Hyde, Cameron J; Elizur, Abigail; Ventura, Tomer https://research.usc.edu.au/discovery/delivery/61USC_INST:ResearchRepository/12126573000002621?l#13127277830002621

Hyde, C. J., Elizur, A., & Ventura, T. (2019). The crustacean ecdysone cassette: a gatekeeper for molt and metamorphosis. Journal of Steroid Biochemistry and Molecular Biology, 185, 172–183. https://doi.org/10.1016/j.jsbmb.2018.08.012 Document Type: Accepted Version

Link to Published Version: https://doi.org/10.1016/j.jsbmb.2018.08.012

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Title: The crustacean ecdysone cassette: A gatekeeper for molt and metamorphosis

Authors: Cameron J. Hyde, Abigail Elizur, Tomer Ventura

PII: S0960-0760(18)30265-6 DOI: https://doi.org/10.1016/j.jsbmb.2018.08.012 Reference: SBMB 5202

To appear in: Journal of Steroid Biochemistry & Molecular Biology

Received date: 10-5-2018 Revised date: 21-8-2018 Accepted date: 25-8-2018

Please cite this article as: Hyde CJ, Elizur A, Ventura T, The crustacean ecdysone cassette: A gatekeeper for molt and metamorphosis, Journal of Steroid Biochemistry and Molecular Biology (2018), https://doi.org/10.1016/j.jsbmb.2018.08.012

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Running title: Endocrinology of crustacean metamorphosis

Cameron J. Hyde, Abigail Elizur and Tomer Ventura

Genecology Research Centre, Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, 4 Locked Bag, Maroochydore, Queensland 4558, Australia

Graphical abstract

Highlights:

 Metamorphosis regulation with emphasis on crustaceans is reviewed ACCEPTED Specific attention is given to the ecdysone MANUSCRIPT pathway and associated nuclear receptors  Gaps in knowledge are identified and future directions are suggested

Abstract

Arthropods have long been utilized as models to explore molecular function, and the findings derived from them can be applied throughout metazoa, including as a basis for medical research.

This has led to the adoption of many representative insect models beyond Drosophila, as each lends its own unique perspective to questions in endocrinology and genetics. However, non- insect arthropods are yet to be realised for the potential insight they may provide in such studies.

The Crustacea are among the most ancient arthropods from which insects descended, comprising a huge variety of life histories and ecological roles. Of the events in a typical crustacean development, metamorphosis is perhaps the most ubiquitous, challenging and highly studied.

Despite this, our knowledge of the endocrinology which underpins metamorphosis is rudimentary at best; although several key molecules have been identified and studied in depth, the link between them is quite nebulous and leans heavily on well-explored insect models, which diverged from the Pancrustacea over 450 million years ago. As omics technologies become increasingly accessible, they bring the prospect of explorative molecular research which will allow us to uncover components and pathways unique to crustaceans. This review reconciles known components of crustacean metamorphosis and reflects on our findings in insects to outline a future search space, with focus given to the ecdysone cascade. To expand our knowledge of this ubiquitous endocrine system not only aids in our understanding of crustacean metamorphosis, but also provides a deeper insight into the adaptive capacity of arthropods throughout evolution.

Keywords: Arthropod, Ecdysone, Endocrine, Methyl farnesoate, Molt, Nuclear receptor

ACCEPTED MANUSCRIPT

Table of Contents

Introduction ...... 4

Endocrinology of molting ...... 9

Endocrinology of metamorphosis ...... 12

Nuclear receptor function ...... 16

Intercepting the ecdysone cassette ...... 19

Conclusion ...... 23

Acknowledgements ...... 25

References ...... 25

ACCEPTED MANUSCRIPT

1. Introduction

Over the past century, model organisms have emerged as a vital tool for exploring physiology, disease and genetics, providing a platform for insights into biological phenomena which can be applied across taxa. The most fruitful metazoan model organism to date is without a doubt

Drosophila melanogaster, whose ease of laboratory rearing and abundance of phenotypes made them an ideal candidate for pioneering exploration of gene function in vivo. As knowledge was transferred to other insect species, complimentary insect models arose to represent the physiological and genetic distinctions between the insect orders, such as holometabolous

(complete metamorphosis) and hemimetabolous (incomplete metamorphosis) development.

This iterative expansion of knowledge and variety of biological perspectives has cultivated a very comprehensive understanding of endocrine pathways and corresponding genes in the insects. The wealth of information accumulated in the insects has provided non-insect researchers with a valuable reference to guide them through novel ventures which would otherwise have been impossible, and a convention arose whereby unexplored species are systematically unravelled through comparison with an appropriate model. This approach is particularly amenable to genetic and endocrine applications, since analogous components are often shared among classes and even across phyla, but its utility is generally limited to the transfer of only fundamental concepts, which must be drawn out and contextualized in the species of interest. For example, a conserved enzyme likely binds a similar substrate to its homolog, but gene expression, specificity and binding kinetics are likely to be different.

Phylogenetic analysis has found the Hexapod clade to be an ancient divergent of the

Pancrustacea and therefore, evolutionarily speaking, insects can be considered as derived crustaceansACCEPTED [1, 2]. It is surprising then to find thatMANUSCRIPT our knowledge of crustacean endocrine regulation is comparatively sparse, and though much has been inferred from insect models there are frequent anomalies which cannot be explained by these distant relatives. Of course, the advent of ecdysis (molting) long predates the divergence of the Pancrustacean ancestor, and

4 thus the mechanism for molting is common to insects and crustaceans [1, 3]. It is well documented that arthropod molting is regulated by cyclical pulses of the steroid hormone ecdysone [4]. Downstream of the ecdysone receptor, a vast suite of genes are upregulated; the resulting transcriptomic shift coordinates cuticle release, osmotic flux, tissue remodelling and the many other physiological processes which orchestrate the molt. This widespread flux of gene expression is orchestrated by the ecdysone cassette (or ecdysone cascade), and involves many members of the nuclear receptor superfamily, including the ecdysone receptor itself, and its accurate profiling in Drosophila has provided significantly to our understanding of nuclear receptor function [5-9]. Our knowledge of the ecdysone cassette and the NR superfamily in crustaceans is comparatively limited with very few studies making a comprehensive curation of the NR family [10], which in Daphnia (Daphniidae) has been shown to comprise at least 25 genes [11]. Holistic characterisation of ecdysone response components could provide interesting and useful perspectives on steroid hormone function and nuclear receptor interactions, particularly in the context of metamorphosis. In this critical transformation, the ecdysone cassette is modified to instigate organism-wide tissue restructuring as the molt unfolds, and the animal which emerges from its spent cuticle often looks remarkably different than before [12, 13]. Although this adaptation of the ecdysone cassette has been well explained in insect models, our understanding in crustaceans has not been well resolved by comparative approaches, perhaps due to the nature of the transformation in these distant relatives, and the lack of genetic and genomic resources in the crustacean field.

A clear distinction between metamorphosis in insects and crustaceans is apparent from the surface; insect metamorphosis takes place gradually during a pronounced pupal phase (hoACCEPTEDlometaboly) or is drawn out across a series MANUSCRIPTof molts (hemimetaboly), whereas in crustaceans the most striking transitions can occur over a single molt, as in the case of crab and lobster larvae, or may be abbreviated within the embryonic development, as in the case of penaeid shrimps. Crustacean metamorphosis is further complicated by the widespread occurrence of serial metamorphoses in the larval stages. The latter is of particular significance

5 since metamorphosis in insects tends to coincide with reproductive development, as in all cases of holometaboly [14-17]. However, metamorphic transition between larval stages (as occurs frequently in crustaceans [18]) requires an uncoupling of metamorphosis from reproductive development, and therefore a segregation of the mechanisms which drive these developmental processes. Such stark physiological disparities highlight potential limitations of transferring knowledge from model organisms [19]. Of course, the limitations imposed by model inference is nothing new, but a lack of resources in the crustacean field has precluded the making of refined, tangible conclusions from this approach. While it has been quite possible to locate and study homologous genes in crustaceans, there are some whose function is found to be quite distinct to that of insects, thereby preventing the conception of coherent pathways. Omics approaches have elucidated many conserved pathways in crustaceans, and indeed every pathway mentioned in this article owes its comprehensiveness to the increasing wealth of next- generation sequencing (NGS) data that crustacean researchers have at their disposal [20-26]. As of 03/05/2018, NCBI’s BioProject database returns 213 crustacean transcriptome entries comprising 2764 sequence read archives, 33% of which are from decapods and 92% of which were published since 2015 (Search criteria: "Crustacea"[orgn] AND "transcriptome gene expression"[Filter] AND "org invertebrate"[Filter]) [27]. Despite the abundance of insect reference data (and several “emerging” model crustaceans), crustacean NGS assemblies are still poorly described, often with less than 30% of transcripts being annotated by any protein database [28-32]. This suggests that the majority of crustacean genes remain undefined, serving hitherto unknown functions and likely accounting for the frequent missing links in crustacean molecular pathways. This highlights the importance of curating and reforming gene families in crustaceans, a process for which NGS data has been exceedingly fruitful. As an example, transcriptomeACCEPTED analysis in the spiny lobster Sagmariasus MANUSCRIPT verreauxi enabled curation of the G- protein coupled receptor (GPCR) gene family by domain prediction, where 50% of rhodopsin- like GPCRs did not have a hypothesised function [33]. In Daphnia pulex, a more comprehensive approach to curating the nuclear receptor (NR) family led to the discovery of the HR97 group of

NRs [11, 34]. Studies of this nature reveal a vast potential for novel discovery which would seldom have been realised by previous methods of ortholog-oriented searching. By elucidating the roles of these undefined genes, we can expand our understanding of gene function and evolutionary history, which informs not only our understanding of crustacean biology but also molecular endocrinology in general. The ecdysone pathway appears to be highly lucrative for this purpose, with endogenous and exogenous cues being interpreted by a suite of neuropeptides, which in turn regulate cyclical release of steroid hormones, eliciting widespread transcriptome modulation through a cascade of NR and transcription factor activity.

The prevailing hypothesis in crustaceans is that metamorphosis is initiated by interaction between ecdysone and a sesquiterpenoid hormone, methyl farnesoate (MF). This hypothesis leans heavily on our comprehensive understanding of insect endocrinology, where juvenile hormone (JH, the insect analog of MF) inhibits metamorphosis to the adult stage. In this model of regulation, an ecdysone peak follows a decline in JH titre to initiate metamorphosis [15].

However, comparative studies which have sought to validate this model in crustaceans have indicated that the mechanism may be more complex. Although MF appears to contribute to larval development in crustaceans, its relation to metamorphic timing is unclear [35].

In order to expand our knowledge of the ecdysone cascade into the Crustacea it is necessary to pursue new perspectives due to the apparent limitations in transferring our knowledge of hexapods, whose divergence from the Pancrustacea occurred over 450 million years ago, approximately coinciding with the appearance of vascular plants on land [2, 36]. However, there are similarities which can certainly be taken advantage of with great effect; one such example is theACCEPTED regulation of molting by the ecdysone cassette. MANUSCRIPT In insects, a clear model of ecdysone-JH interplay emerged quite readily after the discovery and curation of molecular components associated with metamorphosis, particularly the suite of genes that are activated in the ecdysone cascade. In this event, a great number of NRs and transcription factors are upregulated by a peak in ecdysone, translating a simple hormonal

7 signal into wide-scale transcriptomic shift [5]. In noting the components of this cascade which correlate with insect metamorphosis, it became clear which molecules orchestrate the interplay between the ecdysone and JH pathways, and the level at which metamorphosis is controlled in the insects [37-39].

However, our knowledge of the ecdysone cassette in crustaceans is quite limited, and therefore an analogous model eludes definition. Recent advances in sequencing technologies and bioinformatics tools are bringing non-model researchers to the frontier of molecular exploration; the grounds for discovery in these species has never been more fertile, and should be taken advantage of to fuel research and hypotheses of a more explorative nature. Under this context, the objective of this review is to reconcile current knowledge on crustacean metamorphosis and endocrinology, with focus on the nature of NRs and transcription factors as key regulators in the ecdysone cascade, and potential gatekeepers for the unidentified mechanism of metamorphosis regulation in crustaceans.

2. Endocrinology of molting

Arthropods undergo molting (known also as ecdysis) in response to cyclic peaks of the steroid hormone ecdysone. In insects, the neuropeptide prothoracicotropic hormone (PTTH) is released from the Corpus allatum to stimulate ecdysone synthesis in the prothoracic gland [40,

41]; in crustaceans ecdysone synthesis occurs in the Y-organ, located behind the mandibles, and is controlled by an inhibitory mechanism implemented by molt-inhibiting hormone (MIH) neuropeptides, secreted from the X-organ/sinus gland complex in the eyestalk [42-44]. MIH is a memberACCEPTED of the Crustacean Hyperglycaemic Hormone MANUSCRIPT (CHH) family, a number of whose members have been implicated in molt regulation [45]. This was undoubtedly the first endocrine pathway encountered in crustacean research, as its mechanism can be illustrated by simply amputating the eyestalks, a procedure known as ablation or “destalking” [35]. By

8 removing the principal regulator of molt, individuals can be seen to depart from their endogenous molt cycle as molt inhibition has been lifted [46]. Interestingly, multiple-leg autotomy advances the molting of crabs, perhaps due to shared pathways between molt regulation and limb regeneration [47, 48].

This removal of inhibition which has been taken advantage of to shorten the lifecycle of

Penaeids in aquaculture, and ablation is still the most reliable method of promoting maturation in the broodstock. Complete removal of a central endocrine organ is of course incredibly detrimental to the animal’s health and the life expectancy of the broodstock is therefore very short. Our knowledge of crustacean neuropeptides has since expanded well outside the CHH family, revealing orthologs of insect hormones as well as novel hormones whose functions are currently being examined [49] and at least some of these new peptide hormones show promise in expanding our understanding of molting. In insects, bursicon is known to regulate tanning of the new cuticle after a molt, a function which appears to be conserved across arthropods.

Although the cuticle-tanning function has certainly been retained, the crustacean bursicon may possess distinct accessory functions [50, 51]. Another finding of interest is crustacean cardioactive peptide (CCAP), which was first discovered in crustaceans in the 1980s before its insect ortholog became apparent [52]; CCAP has since been found across Arthropoda, and appears to be ubiquitous in its role in molting. A large surge of CCAP during the ecdysis program causes a number of behavioural and physiological responses, including increased cardiovascular activity, which facilitates the molt [53]. Though there are many more neurohormones which exhibit some association with the molting process, regulation of ecdysteroid synthesis is predominantly attributed to the MIH family of neuropeptides [44]. The transmissionACCEPTED of neuropeptide signalling within MANUSCRIPT the Y-organ has also been subject of recent investigation. In insects, it has been demonstrated that ecdysone synthesis in the prothoracic gland is dependent on mTOR signalling pathway, though the exact mechanism is unclear [54,

55]. The mTOR pathway is a known metabolic regulator in mammals, which integrates multiple environmental signals to regulate growth in response to nutritional status [56]. Transcriptomic

9 analyses of the crustacean Y-organ has indicated a conserved role of mTOR with that observed in insect models, where stimulation of ecdysteroid synthesis appears to be reliant on the mTOR signalling [57]. Following eyestalk ablation, a steep rise in ecdysone titre is expected as neuropeptide inhibition of the Y-organ is released [44]. However, suppression of the mTOR pathway was shown to mitigate this effect, indicating that the transmission of neuropeptide signalling to ecdysteroid synthesis is mediated, directly or indirectly, by the mTOR pathway

[58].

The synthesis of ecdysone from dietary cholesterol is mediated by a series of enzymes encoded by the “Halloween” gene family (Fig. 1) [44]. Coordinated primarily by cytochrome P450 enzymes, this pathway guides intermediate compounds through a series of oxidation, reduction and hydroxylation reactions which results in the production of ecdysone, which is released into the hemolymph as a prohormone [59, 60]. Circulating ecdysone is then converted to its bioactive derivative, 20-hydroxyecdysone (20-HE), by the peripherally expressed cyp314a1 gene, which encodes the enzyme Shade. This concluding member of the Halloween gene series coordinates tissue-specific ecdysone signalling by local elevation of 20-HE [20, 61, 62]. It should be noted that while the conventional bioactive product of this pathway is 20-HE, there are several compounds which reportedly take this place in crustaceans, including ponasterone-A and 25-deoxyecdysone, and are known collectively as ecdysteroids [44, 63, 64]. For the purpose of clarity, however, we refer only to ecdysone throughout this article. The ecdysone synthesis pathway has been defined relatively well in crustaceans, following review of the cytochrome

P450s and other members of the Halloween genes [20, 44, 65], which recently led to the discovery of the “Shed” gene, transcribing the only known crustacean analogue of the Shade enzyme,ACCEPTED despite being phylogenetically distinct MANUSCRIPT from its insect counterpart [65]. This finding further illustrates the evolutionary disparities between insects and crustaceans, as lineage- specific evolution has led to distinct enzymes performing a similar role in ecdysone synthesis.

Figure 1: Key molecular events in the crustacean ecdysone pathway.

Ecdysone synthesis occurs in the Y-organ and is coordinated by a family of enzymes transcribed by the Halloween gene family. MIH released from the X-organ/sinus gland complex mediates the molt cycle by inhibiting this pathway, acting on the Y-organ through an unidentified membrane receptor (purple oblong). The initial reactions in ecdysone synthesis have become known as the “Black box” due to their persisting obscurity. Subsequent reactions are catalysed by a series of cytochrome P450 enzymes (green ovals). Ecdysone released into the hemolymph diffuses through cell membranes where it is converted to 20-HE by the final enzyme in the series, Shed. This enables 20-HE to bind the EcR-RXR heterodimer complex which can then locate to ecdysone response elements throughout the genome, recruiting coactivator or corepressor molecules upon binding to modify transcription of the target genes.

Upon reaching its target cells, 20-HE permeates the cell membrane and binds ecdysone receptor

(EcR), a prominent member of the NR superfamily [5, 8, 66]. In order to receive 20-HE as a ligand, the EcR must form a heterodimer with a second NR, the retinoid-X receptor (RXR). The activated EcR-RXR receptor complex then initiates a wide-scale transcriptomic response through the binding and activation of target genes, by recognition of specific hormone response elementsACCEPTED (HREs). In the salivary glands of Drosophila MANUSCRIPT (Drosophila melanogaster, Drosophilidae) larvae, the downstream effects of the ecdysone response can be seen in giant polytene chromosomes, where temporal “puffs” occur over some 340 specific loci, in which EcR-mediated transcription actively unfolds via chromatin remodelling [67-69]. From these observations, a

11 refined suite of “early genes” was identified, encoding the transcription factors Broad and E74, and the NR E75 which then further proliferate downstream transcription of the “late genes” which continue to differentiate the ecdysone signalling pathway [70, 71].

The transcriptomic outcome of an ecdysone response can alternate depending on the endocrine context. At one or more defined stages during development the ecdysone response may induce metamorphosis, which in crustaceans can be regarded as a molt accompanied by broad morphological change. This transformation allows physiological and morphological adaptation to the variety of demands encountered throughout development, and constitutes a remarkable display of gene regulation in that two or more discrete phenotypes are produced from a single genome.

3. Endocrinology of Metamorphosis

The distinction between metamorphic and non-metamorphic molt lies in the presence of JH, which in insects functions to perpetuate the larval phase. Control of metamorphosis by a hemolymph-borne factor was first indicated by experiments involving insect larvae parabiosis, an experimental technique where the circulatory system of two animals is surgically joined. By grafting parts of a 4th instar nymph onto a metamorphic nymph it was shown that metamorphosis could be inhibited, thereby extending larval phase past its natural duration [72,

73]. Thus, an ecdysone peak will only provide the signal for a fully metamorphic molt under reduced JH titre. The role of JH in arthropods has been shown to extend far beyond metamorphosis and, in conjunction with ecdysone, it has adopted multiple roles in the coordination of development including vitellogenesis and reproduction [74-77]. JH comprises a groupACCEPTED of sesquiterpenoid hormones, with JH III MANUSCRIPT being the most prominent in insects [78]. Production of JH III occurs in the corpus allatum in insects, where its synthesis from farnesoic acid is regulated by allatostatins [79, 80]. However, detection of JH III has not been reported in crustaceans; instead its metabolic precursor, MF, has been widely regarded as the functional crustacean JH since its discovery by Laufer et al. in 1987 [35, 81, 82]. Synthesis of MF in

12 crustaceans is thought to take place in the mandibular organ, a gland unique to crustaceans which is considered to be analogous to the insect corpus allatum [20, 83]. The final, rate-limiting step of MF synthesis is the conversion of farnesoic acid to MF, a reaction which is conventionally thought to be catalysed by farnesoic acid O-methyltransferase (FAMeT) [84, 85]. However, the recent discovery of a crustacean juvenile hormone acid O-methyltransferase (JHAMT) ortholog

[20, 86, 87] may cast doubt over current opinion on JH evolution across the Pancrustacea, since

JH synthesis is conventionally thought to be unique to the insects. What JHAMT and FAMeT share, however, is their potential as a rate-limiting step in MF synthesis from the mandibular organ [85, 88], since regulation of MF synthesis typically occurs through the downregulation of

JHAMT or FAMeT [35]. In contrast with insects, crustaceans possess an inhibitory mechanism for the regulation of MF mediated by mandibular organ-inhibiting hormones (MOIHs), cryptic members of the CHH-like neuropeptides secreted from the X-organ/sinus gland complex [35].

Although various MOIH peptides have been characterised in decapods [83, 89], an associated receptor awaits verification. Neuropeptide control of the mandibular organ is highly relevant to our understanding of metamorphosis and reproductive development, since it likely serves as a bottleneck resolving the many complex cues which coordinate development.

It is well established that JH serves as a metamorphic inhibitor in insects [82, 90-92], and it is tempting to propose that crustaceans possess an analogous system. However, support for this inviting hypothesis has proven elusive. Administration of MF to the freshwater prawn

Macrobrachium rosenbergii (Palaemonidae) indicated an inhibitory effect on metamorphosis

[93]. In some shrimps and barnacles, however, administration of MF has been shown to accelerate metamorphosis in late-stage shrimp and barnacle larvae [35, 94-96]; thus the role of MFACCEPTED in regulating crustacean metamorphosis is uncMANUSCRIPTlear, although its hormonal role in reproductive maturation is firmly established [35, 77, 97]. Experiments in the larvae of the crabs Rhithropanopeus harrisii (Panopeidae) and Callinectes sapidus (Portunidae) indicated a role of the sinus gland in morphogenesis; eyestalk ablation of intermediate larvae led to failure or impedance of metamorphosis to the megalopa stage [98]. These results were later

13 corroborated by studies in other decapod species, including Homarus americanus

(Nephropidae) and Portunus trituberculatus (Portunidae) [99, 100]. The impact of eyestalk ablation on metamorphosis was later shown to be accompanied by a rise in MF titres, confirming that these observations were potentially the result of inhibition by MF [101].

Accumulative evidence supports a JH-like function of MF in metamorphosis, where the secretion of MF is suppressed by MOIH to release metamorphic inhibition. Conversely, a transcriptome study of metamorphosis in Sagmariasus verreauxi (Palinuridae) has suggested that while this theory holds true for the puerulus-juvenile metamorphosis, the phyllosoma-puerulus metamorphosis is preceded by sustained FAMeT expression [102]. Although it is possible that

FAMeT expression does not accurately reflect MF titre in this study, these results could suggest that MF does not regulate phyllosoma metamorphosis through the conventional inhibitory mechanism.

The emergence of the CYP15A1 enzyme (which converts MF to JH III) in the insects is thought to have led to the appropriation of MF’s ancestral role to JH III. It is therefore likely that such an ancestral role would remain in crustaceans, though the extent of its functional divergence is unclear [90]. It was recently suggested that MF might have a conserved hormonal role in insects, after it was found to be circulating in the hemolymph of several hexapod species [103].

MF was subsequently suggested as a regulatory component in the metamorphosis of

Drosophila, after finding that topical administration of MF could compensate for compromised

JH synthesis in the larvae [104], further suggesting a conserved function of the ancestral hormone. Conversely, the discovery of cyp15a1 in decapod species raises the question of JH activity in crustaceans, though it is likely that in crustaceans the enzyme functions as part of the MFACCEPTED degradation pathway [28, 65]. MANUSCRIPT The search for an insect JH receptor persisted for well over a decade, with some researchers considering Ultraspiracle (USP; the insect ortholog of RXR) a likely candidate - an alluring hypothesis since JH would be placed in convenient proximity to the EcR-USP receptor complex

[6, 105, 106]. However, the endorsement of USP as a receptor for JH has been overshadowed by the unearthing of a more likely candidate, Methoprene-Tolerant (Met). Met is part of a family of transcription factors known as the basic helix-loop-helix Per-ARNT-Sim (bHLH-PAS) proteins

[107], and found its name after mutant alleles were identified as the source of resistance in

Drosophila to the insecticide methoprene, a JH analogue [108]. The function of Met, like NRs, typically involves DNA-binding as heterodimer complex with its partner, steroid receptor coactivator (SRC) [90, 109]. Subsequent studies drew attention to its role in coordinating metamorphosis, where Met knockdown resulted in premature metamorphosis in insects [39,

91, 110, 111] and Met was subsequently confirmed as a receptor for JH [112].

Following its identification in insects, attention shortly turned towards Met as a receptor for MF in crustaceans. Met homologs were found in two Daphnia species, and receptor assays confirmed similar function to that described in insects, with JH binding promoting transcription of a reporter gene [113, 114]. Furthermore, the Daphnia Met appeared to bind MF with greater affinity than JH III, perhaps suggesting that it functioned as a MF receptor before the adoption of

JH III in the insects [114, 115]. MF is a known sexual determinant in Daphnia magna [116], and analysis of Met expression during this critical period showed concurrent mRNA peaks 24 hours prior to MF sensitivity [113]. Collectively this evidence strongly suggests functional conservation of Met in branchiopods, the class of crustaceans from which insects emerged.

However, due to the close phylogeny of branchiopods and insects, this finding cannot be applied across the Crustacea before verification by further comparative studies. Although Met orthologs have been found in several decapods exhibiting expression patterns consistent with that of a MF receptor [20, 117], its mechanism as a receptor in decapods cannot be confidently asserted withoutACCEPTED experimental demonstration. Daphniid MANUSCRIPT researchers have also characterized a SRC homolog and provided in vitro demonstration of its function as a Met heterodimer partner [113,

114, 118], but it is yet to be confirmed whether this function is conserved across the Crustacea.

An additional hypothesis of MF function is its potential as a ligand for RXR. This theory has been supported by experimental evidence in insects [119, 120], but is somewhat disputed due to

15 binding affinity [121]. Cell-based receptor assays in Daphnia showed that MF did not activate

RXR directly, but did potentiate the activation of EcR-RXR by ecdysteroids in a synergistic manner [122], indicating that MF may be capable of directly modulating the EcR complex. This effect of MF is thought be complementary to the JH-Met axis in insects, but its role is poorly understood [90, 112].

It remains unclear exactly how MF interacts with the ecdysone pathway to modulate downstream transcription. The discovery of a crustacean Met as a potential MF receptor has somewhat narrowed the gap between the MF and ecdysone pathways, since both are now known to culminate in the activation of nuclear transcription factors between which there are feasible, yet little explored links. In light of the potential for MF to intercept the ecdysone pathway at the NR level, it would be imprudent to speculate on models of integration without thoroughly examining the mechanism of NRs.

4. Nuclear receptor function

One can consider NRs as a suite of ligand-mediated transcription factors, which are ubiquitous core elements in endocrine signalling pathways across metazoa [123]. However, there are intricacies in the NR mechanism that this depiction, appropriate as it may be, does not adequately describe. NRs can be attributed to six sub-families based on phylogeny and structure, which are highly conserved across phyla [5]. In Drosophila these families are represented by 21 genes encoding NRs, with EcR being included in subfamily 1 [8]. However, functional divergence has led to expansion of this receptor family in higher organisms; the human genome is currently thought to contain 48 genes encoding NRs, including the thyroid andACCEPTED estrogen receptors [124]. Although key NRs MANUSCRIPT have been isolated and characterised in many crustacean species, gene discovery has been limited to identifying gene homologs [10, 125, 126] and therefore the NR superfamily has not been explored comprehensively in crustaceans.

Despite the extensive expansion of the NR superfamily, a high level of structural conservation exists between vertebrate and invertebrate orthologs, which is exemplified by reports of invertebrate sensitivity to vertebrate hormones [127, 128]. In order to bind DNA and modify transcription, the NR molecule must typically form a dimer; either with a conspecific receptor

(homodimer) or an associate NR (heterodimer). The receptor dimer can then bind a specific motif in the upstream flanking sequence of target genes, while recruiting coactivator or corepressor molecules to facilitate transcriptional modification [129]. Importantly, NRs can also function as transcriptional repressors, particularly in their unliganded state. In this case, ligand binding causes a shift in the NR’s anisotropic (dimer-forming) properties, resulting in the replacement of corepressors with coactivators, thereby promoting transcription [130]. Thus, expression of a particular NR could infer either repression or activation of target genes, depending on the availability of ligands and dimer partners – the implications of these dynamic interactions are demonstrated in Fig. 2. Section A shows a functional heterodimer complex in the absence of a ligand, which results in no transcriptional modification. This may be considered

ACCEPTED MANUSCRIPT

Figure 2: Functional mechanisms of nuclear receptors

The 5 sections above illustrate the transcriptional outcomes that can be elicited by NRs under different molecular conditions. Yellow circles denote a ligand and blue oblongs show its cognate NR. An associate NR is shown by a grey oblong and dimerization is inferred by two NRs which are overlayed. Transcriptional upregulation is denoted by “+” and suppression of transcription by “-”. In section (d), an irregular shape represents a chaperone protein necessary for translocation of the cognate NR to the nucleus.

as the default outcome from low hormone titres, but the subsequent sections illustrate alternative outcomes which contradict this assumption. Transcriptional repression is shown in section (b), where the absence of the cognate receptor causes the associated heterodimer partner to resort to homodimer formation, which induces DNA binding and transcription repression at target sites (note that each dimer configuration induces DNA-binding to a specific motif,ACCEPTED hence each dimer configuration binds to MANUSCRIPTa different site). Conversely, section (c) demonstrates an absence of transcriptional response in the presence of both ligand and cognate receptor, due to the lack of a heterodimer partner. This has been demonstrated in Aedes aegypti

(Culicidae), where a third NR intercepts the ecdysone response by competing with EcR for dimerization with USP [131]. Section (d) shows a positive transcriptional response which is

18 facilitated by translocation of the cognate receptor (which in this case would constitutively reside in the cytoplasm) to the nucleus following ligand-binding. This mechanism introduces the need for a chaperone protein to be present and available. Conventional receptor activation is illustrated in section (e), where the presence of ligand, cognate receptor and heterodimer partner allows transcriptional activation at the target sites.

Dimer interaction between NRs provides the potential for cross-talk between hormonal pathways, particularly in receptors binding multiple dimer partners, a property exemplified by the RXR [123, 124, 132] which is noted for its promiscuity as a heterodimer partner [131, 133,

134]. The RXR is considered to be a vital accessory in many hormonal pathways where it serves as an obligate heterodimer partner to multiple receptors of lipid hormones, including the EcR

[7, 135]. In this context, the dipteran USP has been found to regulate ecdysone responses through competitive dimer formation and expression of alternative USP alleles [136, 137], thereby indicating the capability of USP in serving a regulatory (as opposed to facilitative) role in endocrine pathways. In crustaceans, similar studies have revealed alternative mRNA splicing in both USP and EcR transcripts, which potentially function to tailor the ecdysone response to specific tissues [138-141]. Splice isoforms are well documented in the insect EcR [142,

143];[144], so their discovery in crustaceans does not come as a surprise. However, insects are usually found to have only two EcR splice variants, whereas crustaceans have been found with anything from two to eight variants [145], perhaps indicating that the crustacean EcR has experienced a greater degree of functional divergence. There have been several studies which examine the functional significance of EcR and RXR splice variants in crustaceans. Four EcR splice variants have been found in the Chinese mitten crab Eriocheir sinensis (Varunidae), with oneACCEPTED isoform expressed specifically in the testes MANUSCRIPT [146] and another in the ovary [145]. A third EcR variant was found to fluctuate in expression with molt cycle, and RNA silencing of this isoform was found to delay molting by a short but significant period [145]. Eyestalk ablation, which typically causes an increase in ecdysteroid titre [44, 147], resulted in the downregulation of one EcR variant but the upregulation of another, indicating differential regulation of mRNA

19 splicing by feedback mechanisms [146]. Five RXR variants found in the Oriental freshwater prawn Macrobrachium nipponense (Palaemonidae) were differentially expressed across tissues, with one isoform being specific to the ovary [148]. In the land crab Gecarcinus lateralis

(Gecarcinidae), the RXR was found to encode nine variants with differential expression between thoracic and claw muscle, and the highest variant diversity found in the gonad, which perhaps reflects the more complex endocrine regulation found in these tissues [139]. Aside from the higher diversity of splice variants found in crustaceans, there is also a structural distinction to those found in the insect EcR. Splice junctions in the insect EcR are generally located in the A/B domain, but splice variants in crustaceans frequently occur in the LBD (as reviewed by Chen et al. [145]), which can potentially alter the receptor’s dimerization properties, ligand-binding affinity and even DNA-binding properties. In the fiddler crab Uca pugilator (Ocypodidae), examination of EcR and RXR isoforms by glutathione S-transferase (GST) pull-down experiments identified differential HRE binding by the EcR-RXR complex, caused by a 33-AA insertion in the LBD of the RXR which facilitated dimerization with EcR [63]. Conversely, a more recent study utilizing surface plasmon resonance (SPR) found the opposite relationship, with dimerization occurring only with the absence of the 33-AA insertion [149]. This could perhaps be explained by differences in methodology between experiments, which should perhaps be verified by future studies. Splicing of the hinge region has also been found in the RXR and EcR

[63, 138] and can hypothetically affect HRE-binding [150, 151]. Functional characterization of the U. pugilator RXR showed that hinge region alternative splicing did not impede dimerization with EcR [63], indicating that splicing in this region may result in several viable EcR/RXR complexes with differential signal transduction. However,ACCEPTED ecdysteroid binding to the EcR-RXR complexMANUSCRIPT was not shown to enhance DNA-binding (although DNA-binding was enhanced as expected with a Drosophila EcR) in electromobility shift assays [152], which likely indicates that there are aspects of this mechanism that are not fully understood in crustaceans. This observation may relate to EcR and RXR splice-variant diversity, since isoform pairing may be critical to ligand-mediated signal transduction. This

20 introduces an additional layer of complexity in NR signal transduction mechanisms (in addition to those described in figure 2) and highlights the challenge of representing biological function with in vitro assays. These findings demonstrate that understanding allosteric reactions between receptors is vital to provide molecular context to our observations of NRs in an endocrine setting. In crustaceans, splice variants and competitive dimerization should be further explored by cell-based receptor assays [64, 149, 153] to better clarify the conditions that attenuate or potentiate specific DNA-binding responses. Elucidating the expression of additional crustacean NRs during ecdysone release will improve our understanding of these interactions and the individual roles of NRs during molt and metamorphosis.

5. Intercepting the ecdysone cassette

Since coordination of molting by the ecdysone cassette is common to both insects and crustaceans [154], it is highly likely that metamorphosis in crustaceans is determined by modulating one or more ecdysone-responsive genes. Our current understanding of crustacean metamorphosis is based on the assumption that MF serves as a metamorphic inhibitor, though the mechanism which links MF to the ecdysone cassette is currently unknown. A logical approach to solving this knowledge gap is to examine and characterise the molecular response downstream of MF.

In the insects, a compelling link between the JH and ecdysone pathways has accumulated through a series of experimental studies. The discovery of Met as a functional JH receptor has been a significant milestone in our understanding of arthropod endocrine systems, which has allowed researchers to define specific JH responsive genes. Of particular interest is a pathway connectingACCEPTED Met to the ecdysone-responsive genes MANUSCRIPT broad (known also as broad-complex) and E93, both of which encode transcription factors [155-157]. Broad is a prominent member of the broad/tramtrack/bric-a-brac (BTB) family of transcription factors, a prolific yet little-studied group of proteins which share a highly conserved BTB domain, and typically feature one or several zinc-finger motifs which form a DNA-binding domain. The BTB domain itself functions

21 as a dimer interface, allowing the protein to interact with associated transcription factors and coactivator molecules. The Drosophila genome is thought to encode 85 BTB proteins [158], but this number varies greatly between phyla and their function has been scarcely explored outside of model species, where a role in metamorphosis has been identified [159].

Drosophila larvae carrying a mutant broad allele developed competently until the final larval stage when they failed to metamorphose [160], which indicates that Broad facilitates a metamorphic pupal molt in the ecdysone cascade (Fig. 3). Subsequent evidence supports Broad as a regulator of metamorphosis, though some functional disparities may exist between dipteran and coleopteran species [157, 161-164]. However, the link to JH did not become apparent until the identification of Krüppel-homolog 1 (Kr-h1) as a mediator between Met and

Broad in Drosophila [39, 111]. In vitro application of endogenous JHs to Bombyx mori

(Bombycidae) cells resulted in rapid upregulation of Kr-h1, while excision of the corpus allatum in larvae reduced Kr-h1 mRNA to trace levels. Subsequently, a putative JH-responsive element was identified in the genome upstream of Kr-h1 [165]. These findings were supplemented by

RNAi knockdown experiments in Tribolium castaneum (Tenebrionidae), which demonstrated inhibition of metamorphosis by Kr-h1, through repression of Broad [39, 111] (Fig. 3).

In addition to Broad, the transcription factor E93 was identified as a key coordinator of metamorphosis [166, 167], which was initially suggested to act upstream of Broad by downregulating Kr-h1 expression [17]. However, a parallel study concluded that Kr-h1 serves as an upstream regulator of E93, promoting development towards the adult physiology [16, 37].

Accumulated evidence places Broad and E93 as central elements in the bridge between JH and metamorphosis in insects, both regulated directly by Kr-h1, with the general consensus that BroadACCEPTED functions as a pupal specifier and E93 asMANUSCRIPT an adult specifier during the process. However, the distinction between the roles in different species requires clarification [17, 90, 164], and it can only be speculated how such a mechanism might function in crustaceans. A crustacean Kr- h1 was first identified in the mud crab Scylla paramamosain (Portunidae), and though MF

22 administration caused an upregulation of Met, there was no associated response in Kr-h1 expression [126]. A more recent study of Daphnia also indicates a lack of MF-responsiveness, as well as MF-independent function during embryogenesis [168], which suggests that a significant degree of functional divergence between daphniids and insects. Further studies should aim to characterise Kr-h1 in other crustaceans to clarify whether these observations hold true across the crustacean, in which case the canonical insect JH pathway must have arisen after divergence from the Pancrustacean ancestor.

Although the MF-responsive genes have been little explored in crustaceans, several key ecdysone-responsive genes have already been identified and characterised. Most widely studied is the early-response gene E75, which many studies have shown to express differentially throughout the molt cycle and larval development in a wide range of tissues such as Y-organ, muscle, epidermis, hepatopancreas, eyestalk and nerve cord [169-172]. In vivo studies have shown that E75 is upregulated by ecdysone administration [172, 173] and eyestalk ablation

[169], and promotes transcription of tissue-specific genes in the epidermis and hepatopancreas during the molt, relating to processes such as cuticle remodelling and protein catabolism [173].

RNAi knockdown of E75 in the Chinese white shrimp Fenneropenaeus chinesensis (Penaeidae) impeded retraction of the epidermis and development of new cuticle, leading to molting lethality. The same treatment in P. trituberculatus was shown to reduce MIH expression in the eyestalk, indicating that E75 may relay negative feedback from the ecdysone cassette to stabilize neuropeptide regulation following ecdysone release [169]. Our understanding of the crustacean E75 remains quite rudimentary, and further study will certainly reveal more about the pathways that are under its regulation during molting and metamorphosis. The Drosophila E75ACCEPTED is thought to bind metabolites such as nitric MANUSCRIPT oxide, which regulates dimerization with its partner, HR3 [174]. This mechanism has not yet been inspected in a crustacean species, but HR3 itself has been characterised in several species. Ecdysteroid administration in H. americanus resulted in upregulation of HR3 in the muscle and epidermis, but downregulation in the eyestalk, which may indicate a role in negative feedback, perhaps synergistically with E75 as a

23 heterodimer [175]. In Daphnia, HR3 was upregulated 30-fold from basal levels by 20-HE administration, while effect on E75 transcription was minimal [176]. Several other ecdysone- responsive NRs have been suggested by gene expression analysis of decapods, including E74,

Ftz-F1, ERR, HR38 and E78 [171, 177]. For the study of metamorphosis, perhaps the most interesting ecdysone-response gene to elucidate is a crustacean ortholog of Broad which, to our knowledge, has been characterised only briefly in P. mondon. In these studies, a gene encoding a

BTB protein with high similarity to Broad-complex isoform in Apis melifera (Apidae) was found to express at highest levels in the ovary, and constitutively in other tissues, and was significantly upregulated by eyestalk ablation during vitellogenesis [178, 179].

These findings demonstrate that various members of the ecdysone cassette are well conserved across the Pancrustacea, although their specific roles in molting and metamorphosis merit further investigation, potentially through appropriate experimental design in combination with

Omics approaches, to allow a thorough comparison against the insect pathways previously described. As suggested earlier, however, a key distinction between crustacean and insect metamorphosis is the uncoupling of reproductive maturation from the metamorphic molt, as well as the lack of the pupal phase which is seen in holometabolous insects. Therefore, it is reasonable to predict that some components of metamorphosis determination are not shared between insects and crustaceans, and that a novel pathway links MF to the ecdysone cassette – a hypothesis which so far complements the apparent divergence of Kr-h1 function in crustaceans

[126, 168]. In this sense, it is likely that crustacean research will help to clarify the endocrine determination of metamorphosis and reproductive development by taking advantage of the temporal segregation of these processes in crustaceans. ACCEPTED MANUSCRIPT

Based on developments in insect models, the most promising avenue to advance our understanding of metamorphosis in crustaceans lies in the association between MF and the ecdysone pathway, for which several leading hypotheses and potential mechanisms have been discussed (Fig. 3). Key components in these pathways have been highlighted, including ecdysone, MF and JH III as hormonal ligands, and EcR, RXR and Met as their prospective receptors, which interact to initiate or inhibit metamorphosis while also coordinating the molt cycle. Investigating crustacean orthologs of these genes may reveal similar links between these pathways that give rise to a model of metamorphosis regulation, which could lead us towards a better understanding of larval development, while exhibiting novel mechanisms of steroid- directed development. The identification of downstream responses to MF would be highly relevant to such applications, particularly those which specify the onset of metamorphosis. An important consideration is that in crustacean metamorphosis regulation, the insect roles of Kr- h1, Broad, and E93 may have been adopted by novel MF-regulated genes, particularly considering the absence of a crustacean E93. Given the omnipresence of NRs as key components

Figure 3: Models of metamorphosis regulation in insects and crustaceans

The diagrams illustrate the interactions between the juvenile hormone (JH) and ecdysone pathways, which are thought to regulate insect metamorphosis and currently form a basis for hypotheses in crustaceansACCEPTED. Section (a) depicts the outcome of anMANUSCRIPT ecdysone peak in an insect, where the presence of JH (green circles) inhibits metamorphic onset through binding of Methoprene-tolerant (Met) which upregulates Krüppel-homolog 1 (Kr-h1) expression and subsequently suppressed Broad and/or E93. The ecdysone pathway is depicted by 20-hydroxyecdysone (red circles) binding to the Ultraspiracle- Ecdysone receptor complex (blue and red oblongs) which then binds target DNA response motifs. A putative crustacean model is illustrated in section (b), where methyl farnesoate (MF) replaces JH. Hypothesised components are shown as dotted outlines and text in grey.

25 in this regulatory system (Fig. 3), curating and characterising the NR superfamily in crustaceans would be a great asset in the search for novel and perhaps vital components. Moreover, further exploration into the obscure and prolific BTB family in crustaceans will likely reveal hitherto unknown regulators of development, analogous to the insect broad.

6. Conclusion

Several models of metamorphosis regulation have been described which are primarily based on studies of insect models. However, before exploring possibilities in depth and complexity, it should be remembered that there are several key hypotheses to be affirmed in crustaceans, particularly in decapods. The concept of ecdysone as a molt regulator is embedded in studies throughout arthropods, but the exact role of MF in crustaceans is uncertain [35, 93, 180].

Although evidence has strongly suggested an implication in metamorphosis, the mechanism is not as clear as with JH in insects. However, it seems plausible that the function of Met as a JH receptor is conserved between insects and crustaceans, since its function in daphniids indicates that this function arose prior to the Pancrustacean ancestor [113, 118]; in any case, demonstration of a functional MF receptor in decapods would be a great asset to our understanding of the crustacean MF pathway, and could be further contextualized by in vivo experiments during metamorphosis.

Due to their abundance in the ecdysone cascade, and the division of pathways which they govern during ecdysis, the NR superfamily presents a fruitful ground for exploring links to MF regulation. Insect studies have found that the root of metamorphosis regulation lies within the interactions of these complex and powerful transcription regulators, and it is therefore likely thatACCEPTED in crustaceans the decision for a molt to beMANUSCRIPT metamorphic or otherwise will be uncovered by charting the activity of NRs and associated transcription factors as the ecdysone cassette unfolds in vivo. Additionally, transcriptomic studies can be further utilized by profiling expression response following hormonal administration, which would be of great interest for elucidating the ecdysone and MF pathways. However, functional analysis of these pathways

26 presents a great challenge to crustacean researchers, without the molecular tools and laboratory models that facilitate functional studies in insects. Perhaps the most widespread tool for functional studies in crustaceans is RNA interference with double-stranded RNA, which has allowed knockdown of target genes in many penaeids, carideans and brachyurans [48, 169,

181], although susceptibility to this approach is highly variable between species. For investigating the functional roles of EcR and RXR variants, establishing a panel of RNAi assays to specifically target multiple isoforms would allow for functional comparison between splice variants, where current studies have targeted only a single isoform [48, 145], although the short size of splice fragments may prohibit specificity in some cases [63, 138]. With little access to naturally occurring mutant phenotypes, development of CRISPR assays for gene knockdown may present exciting opportunities for crustacean researchers over the next decade [182, 183], with the prospect of introducing customised mutant genetic lines to examine gene function more precisely and over the organism’s lifetime. In the case of NR splice variants, spliced intron regions could be edited to render their products dysfunctional to assess the impact of knockdown on long-term development, particularly those which are specific to the gonad or molt cycle [145, 148], which may underlie the segregated regulation of these processes in crustaceans. Such technologies could be utilized for many other species with appropriate traits to answer specific questions. For the study of metamorphosis regulation, Palinurid lobsters provide a unique insight into molting and metamorphosis with the phyllosoma larva, which allows accurate tracking of molt stage and metamorphosis onset due to a short and consistent intermolt phase, as well as physiological observation permitted by the transparent cuticle and flat body form [19, 102]. Although these animals lend themselves well to studying molt and metamorphosis, the extreme difficulty in culturing them does not predispose them to serve as a generalACCEPTED laboratory model. Alternatively, many freshwaterMANUSCRIPT crayfish (Astacidae, Cambaridae and

Parastacidae) species allow accurate tracking of molt stage by monitoring of gastrolith deposits by X-ray [184], and are an attractive laboratory model due to their ease of rearing. Another species which matches well the traits of a conventional model organism is the cherry shrimp

Neocaridina denticulata (Atyidae) (reviewed by Mykles and Hui [185]), being small, transparent, easily reared and with a draft genome already available [186]. The improvement of decapod genomes in the future will also provide the opportunity to examine molecular pathways from different perspectives; with EcR HRE motifs being well defined in decapods [63,

138] it may be possible to screen promoters in silico for such regulatory elements [187, 188] or alternatively employ a ChIP-seq approach to identify specific targets of the EcR/RXR complex and better characterize the downstream response [189, 190].

The ecdysone cassette presents an attractive system for exploring a central steroid pathway, which has undergone vast evolutionary adaptation and is perhaps central to the expansion of the Arthropoda. The process of molting and metamorphosis has undergone over 450 million years of adaptive divergence to facilitate the great variation in life history found in the arthropods, but our understanding of the underlying endocrine system is largely confined to hexapods and could be greatly expanded and contextualized by examining their evolutionary precursors, the Crustacea. Identifying the molecular adaptations corresponding to such distinct life histories and developmental events will enhance our understanding of transcriptomic regulation by nuclear receptors, and in particular showcase the mechanisms by which such pathways can be augmented to generate alternate phenotypic outcomes. Model insects have played an irreplaceable role in demonstrating molecular mechanisms, developmental pathways and many other fundamental biological principles, and this role will certainly continue into the foreseeable future. However, NGS and omics technologies can now afford us the prospect of distilling such knowledge across the many non-model organisms which are equally relevant, yet vastly under-exploited. The now widespread mining of transcriptome and genome data providesACCEPTED us the opportunity to not only replicate MANUSCRIPT previous findings in insects, but to expand upon and contextualize homologous pathways which have been separated by distinct and prolonged evolutionary histories.

7. Acknowledgements

The authors would like to thank the University of the Sunshine Coast for providing funding to

Cameron Hyde through a USCIRS scholarship. We would also like to acknowledge funding granted to Dr. Tomer Ventura from the Australian Research Council (ARC DP160103320) which supported this research.

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