Molecular characterization of the cytochrome P450 enzyme CYP18A1 in Henosepilachna vigintioctopunctata.

In insects, the expression of 20E response genes that initiate metamorphosis is triggered by a pulse of 20-hydroxyecdysone (20E). The 20E pulse is generated through two processes: synthesis, which increases its level, and inactivation, which decreases its titer. CYP18A1 functions as an ecdysteroid 26-hydroxylase and plays a role in 20E removal in several representative insects. However, applying 20E degradation activity of CYP18A1 to other insects remains a significant challenge. In this study, we discovered high levels of Hvcyp18a1 during the larval and late pupal stages, particularly in the larval epidermis and fat body of Henosepilachna vigintioctopunctata, a damaging Coleopteran pest of potatoes. RNA interference (RNAi) targeting Hvcyp18a1 disrupted the pupation. Approximately 75% of the Hvcyp18a1 RNAi larvae experienced developmental arrest and remained as stunted prepupae. Subsequently, they gradually turned black and eventually died. Among the Hvcyp18a1-depleted animals that successfully pupated, around half became malformed pupae with swollen elytra and hindwings. The emerged adults from these deformed pupae appeared misshapen, with shriveled elytra and hindwings, and were wrapped in the pupal exuviae. Furthermore, RNAi of Hvcyp18a1 increased the expression of a 20E receptor gene (HvEcR) and four 20E response transcripts (HvE75, HvHR3, HvBrC, and HvαFTZ-F1), while decreased the transcription of HvßFTZ-F1. Our findings confirm the vital role of CYP18A1 in the pupation, potentially involved in the degradation of 20E in H. vigintioctopunctata.

Stage-Specific Demographic Effects of Hydrologic Variation in a Stream Salamander.

AbstractWe lack a strong understanding of how organisms with complex life histories respond to climate variation. Many stream-associated species have multistage life histories that are likely to influence the demographic consequences of floods and droughts. However, tracking stage-specific demographic responses requires high-resolution, long-term data that are rare. We used 8 years of capture-recapture data for the headwater stream salamander Gyrinophilus porphyriticus to quantify the effects of flooding and drying magnitude on stage-specific vital rates and population growth. Drying reduced larval recruitment but increased the probability of metamorphosis (i.e., adult recruitment). Flooding reduced adult recruitment but had no effect on larval recruitment. Larval and adult survival declined with flooding but were unaffected by drying. Annual population growth rates (λ) declined with flooding and drying. Lambda also declined over the study period (2012-2021), although mean λ was 1.0 over this period. Our results indicate that G. porphyriticus populations are resilient to hydrologic variation because of compensatory effects on recruitment of larvae versus adults (i.e., reproduction vs. metamorphosis). Complex life cycles may enable this resilience to climate variation by creating opportunities for compensatory demographic responses across stages. However, more frequent and intense hydrologic variation in the latter half of this study contributed to a decline in λ over time, suggesting that increasing environmental variability poses a threat even when demographic compensation occurs.

The embryonic role of juvenile hormone in the firebrat, Thermobia domestica, reveals its function before its involvement in metamorphosis.

To gain insights into how juvenile hormone (JH) came to regulate insect metamorphosis, we studied its function in the ametabolous firebrat, Thermobia domestica. Highest levels of JH occur during late embryogenesis, with only low levels thereafter. Loss-of-function and gain-of-function experiments show that JH acts on embryonic tissues to suppress morphogenesis and cell determination and to promote their terminal differentiation. Similar embryonic actions of JH on hemimetabolous insects with short germ band embryos indicate that JH's embryonic role preceded its derived function as the postembryonic regulator of metamorphosis. The postembryonic expansion of JH function likely followed the evolution of flight. Archaic flying insects were considered to lack metamorphosis because tiny, movable wings were evident on the thoraces of young juveniles and their positive allometric growth eventually allowed them to support flight in late juveniles. Like in Thermobia, we assume that these juveniles lacked JH. However, a postembryonic reappearance of JH during wing morphogenesis in the young juvenile likely redirected wing development to make a wing pad rather than a wing. Maintenance of JH then allowed wing pad growth and its disappearance in the mature juvenile then allowed wing differentiation. Subsequent modification of JH action for hemi- and holometabolous lifestyles are discussed.

Bacterial c-di-GMP signaling gene affects mussel larval metamorphosis through outer membrane vesicles and lipopolysaccharides.

Biofilms serve as crucial cues for settlement and metamorphosis in marine invertebrates. Within bacterial systems, c-di-GMP functions as a pivotal signaling molecule regulating both biofilm formation and dispersion. However, the molecular mechanism of how c-di-GMP modulates biofilm-induced larval metamorphosis remains elusive. Our study reveals that the deletion of a c-di-GMP related gene in Pseudoalteromonas marina led to an increase in the level of bacterial c-di-GMP by knockout technique, and the mutant strain had an enhanced ability to produce more outer membrane vesicles (OMVs) and lipopolysaccharides (LPS). The mutant biofilms had higher induction activity for larval metamorphosis in mussels Mytilus coruscus, and OMVs play a major role in the induction activity. We further explored the function of LPS in OMVs. Extracted LPS induced high larval metamorphosis rate, and LPS content were subject to c-di-GMP and LPS-biosynthesis gene. Thus, we postulate that the impact of c-di-GMP on biofilm-induced metamorphosis is mediated through OMVs and LPS.

Specification of distinct cell types in a sensory-adhesive organ important for metamorphosis in tunicate larvae.

The papillae of tunicate larvae contribute sensory, adhesive, and metamorphosis-regulating functions that are crucial for the biphasic lifestyle of these marine, non-vertebrate chordates. We have identified additional molecular markers for at least 5 distinct cell types in the papillae of the model tunicate Ciona, allowing us to further study the development of these organs. Using tissue-specific CRISPR/Cas9-mediated mutagenesis and other molecular perturbations, we reveal the roles of key transcription factors and signaling pathways that are important for patterning the papilla territory into a highly organized array of different cell types and shapes. We further test the contributions of different transcription factors and cell types to the production of the adhesive glue that allows for larval attachment during settlement, and to the processes of tail retraction and body rotation during metamorphosis. With this study, we continue working towards connecting gene regulation to cellular functions that control the developmental transition between the motile larva and sessile adult of Ciona.

Unraveling the transcriptomic landscape of eye migration and visual adaptations during flatfish metamorphosis.

Flatfish undergo a remarkable metamorphosis from symmetrical pelagic larvae to fully asymmetrical benthic juveniles. The most distinctive features of this transformation is the migration of one eye. The molecular role of thyroid hormone in the metamorphosis process in flatfishes is well established. However, the regulatory network that facilitates eye movement remains enigmatic. This paper presents a morphological investigation of the metamorphic process in turbot eyes, using advanced imaging techniques and a global view of gene expression. The study covers migrant and non-migrant eyes and aims to identify the genes that are active during ocular migration. Our transcriptomic analysis shows a significant up-regulation of immune-related genes. The analysis of eye-specific genes reveals distinct patterns during the metamorphic process. Myosin is highlighted in the non-migrant eye, while ependymin is highlighted in the migrant eye, possibly involved in optic nerve regeneration. Furthermore, a potential association between the alx3 gene and cranial restructuring has been identified. Additionally, it confirmed simultaneous adaptation to low light in both eyes, as described by changes in opsins expression during the metamorphic process. The study also revealed that ocular migration activates systems asynchronously in both eyes, providing insight into multifaceted reorganization processes during metamorphosis of flatfish.

MicroRNA miR-7-5p targets MARK2 to control metamorphosis in Galeruca daurica.

The MARK2 gene, coding microtubule affinity-regulating kinase or serine/threonine protein kinase, is an important modulator in organism microtubule generation and cell polarity. However, its role in the metamorphosis of insects remains unknown. In this study, we found a conserved miRNA, miR-7-5p, which targets MARK2 to participate in the regulation of the larval-pupal metamorphosis in Galeruca daurica. The dual luciferase reporter assay showed that miR-7-5p interacted with the 3' UTR of MARK2 and repressed its expression. The expression profiling of miR-7-5p and MARK2 displayed an opposite trend during the larval-adult development process. In in-vivo experiments, overexpression of miR-7-5p by injecting miR-7-5p agomir in the final instar larvae down-regulated MARK2 and up-regulated main ecdysone signaling pathway genes including E74, E75, ECR, FTZ-F1 and HR3, which was similar to the results from knockdown of MARK2 by RNAi. In contrast, repression of miR-7-5p by injecting miR-7-5p antagomir obtained opposite effects. Notably, both overexpression and repression of miR-7-5p in the final instar larvae caused abnormal molting and high mortality during the larval-pupal transition, and high mortality during the pupal-adult transition. The 20-hydroxyecdysone (20E) injection experiment showed that 20E up-regulated miR-7-5p whereas down-regulated MARK2. This study reveals that the accurate regulation of miRNAs and their target genes is indispensable for insect metamorphosis.

The BTB transcription factor, Abrupt, acts cooperatively with Chronologically inappropriate morphogenesis (Chinmo) to repress metamorphosis and promotes leg regeneration.

Many insects undergo the process of metamorphosis when larval precursor cells begin to differentiate to create the adult body. The larval precursor cells retain stem cell-like properties and contribute to the regenerative ability of larval appendages. Here we demonstrate that two Broad-complex/Tramtrack/Bric-à-brac Zinc-finger (BTB) domain transcription factors, Chronologically inappropriate morphogenesis (Chinmo) and Abrupt (Ab), act cooperatively to repress metamorphosis in the flour beetle, Tribolium castaneum. Knockdown of chinmo led to precocious development of pupal legs and antennae. We show that although topical application of juvenile hormone (JH) prevents the decrease in chinmo expression in the final instar, chinmo and JH act in distinct pathways. Another gene encoding the BTB domain transcription factor, Ab, was also necessary for the suppression of broad (br) expression in T. castaneum in a chinmo RNAi background, and simultaneous knockdown of ab and chinmo led to the precocious onset of metamorphosis. Furthermore, knockdown of ab led to the loss of regenerative potential of larval legs independently of br. In contrast, chinmo knockdown larvae exhibited pupal leg regeneration when a larval leg was ablated. Taken together, our results show that both ab and chinmo are necessary for the maintenance of the larval tissue identity and, apart from its role in repressing br, ab acts as a crucial regulator of larval leg regeneration. Our findings indicate that BTB domain proteins interact in a complex manner to regulate larval and pupal tissue homeostasis.

Environmentally relevant concentrations of aqueous atorvastatin produce alterations in cholesterol biosynthesis and gene expression patterns in Xenopus laevis.

Numerous studies report active pharmaceutical compounds detected in both wastewater effluent and surface waters. Exposure to statin drugs in general, and atorvastatin in particular, is likely to be a concern. We hypothesized that chronic exposure to low concentrations of atorvastatin in water would result in an adverse effect on production of steroids regulating growth and development of the model amphibian Xenopus laevis. The FETAX assay was used to evaluate the effects of a range of doses of atorvastatin on developing embryos. A 60 day metamorphosis assay assessed the effects of aqueous atorvastatin exposure at environmentally concentrations on metamorphosing tadpoles. A 60 day chronic flow-through exposure evaluated the effects of chronic low concentrations of atorvastatin on adults. The purpose of the FETAX assay was to confirm that atorvastatin can reduce circulating cholesterol in X. laevis with a similar manner to that expected in humans. The results of the 60-day flow-through exposure on metamorphosing tadpoles showed significant evidence of altered cholesterol biosynthesis. The dose-dependent increase in cyp19a1 expression also indicated that the steroidogenesis pathway was affected. The RNAseq analysis confirmed that exposure to environmentally relevant concentrations of atorvastatin does cause significant alterations to global transcriptional profiles in a manner consistent with dysregulation of the cholesterol biosynthesis pathway, both through the downregulation of many genes involved in that pathway, but also in the impacts to other, related pathways. The qPCR data for both adult males and adult females indicated only slight changes in expression with the exception that hmgcr was significantly downregulated in males, and cyp3a4 expression was significantly downregulated in females. The data we present here indicated that chronic exposure to environmentally relevant concentrations of atorvastatin does have the potential to impact early life stage frogs, particularly by altering expression of genes involved in critical molecular pathways.

Polymodal sensory perception drives settlement and metamorphosis of Ciona larvae.

The Earth's oceans brim with an incredible diversity of microscopic lifeforms, including motile planktonic larvae, whose survival critically depends on effective dispersal in the water column and subsequent exploration of the seafloor to identify a suitable settlement site. How their nervous systems mediate sensing of diverse multimodal cues remains enigmatic. Here, we uncover that the tunicate Ciona intestinalis larvae employ ectodermal sensory cells to sense various mechanical and chemical cues. Combining whole-brain imaging and chemogenetics, we demonstrate that stimuli encoded at the periphery are sufficient to drive global brain-state changes to promote or impede both larval attachment and metamorphosis behaviors. The ability of C. intestinalis larvae to leverage polymodal sensory perception to support information coding and chemotactile behaviors may explain how marine larvae make complex decisions despite streamlined nervous systems.

Epichordal vertebral column formation in Xenopus laevis.

Although Xenopus Laevis is the most widely used model amphibian, skeletal development of its vertebral column has not been well illustrated so far. The mode of vertebral column development in anurans has been classified into two modes: perichordal and epichordal. Xenopus vertebral column formation is believed to follow the epichordal mode, but this aspect has been underemphasized, and illustrative examples are currently unavailable to the scientific community. This study documents the entire process of vertebral column formation in X. laevis, from the initial neural arch formation to the completion of metamorphosis. These images reveal that the neural arch arises from the dorsal lamina and lateral pedicle primordia, with no strict adherence to an anteroposterior sequence. Unlike other species, Xenopus centrum primordia exclusively form at the expanded ventral margins of neural arches, rather than from the cartilaginous layer surrounding the notochord. These paired centrum primordia then fuse at the ventral midline, dorsal to the notochord, and subsequently the notochord degenerates. This mode of centrum formation differs from the traditional epichordal mode, indicating that Xenopus might have lost the ability to form a cartilaginous layer around the notochord. Instead, the neural arch's ventral margin appears to have evolved to incorporate centrum precursor cells at its base, thereby forming a centrum-like structure compensating for the absence of a true centrum. It is widely accepted that postsacral vertebrae lack centra, only possessing neural arches, and eventually fuse with the hypochord to form the urostyle. However, we have shown that the paired ventral ends of the postsacral vertebrae also fuse at the midline to form a centrum-like structure. This process might extend to the trunk region during centrum formation. In addition to these findings, we offer evolutionary insights into the reasons why Xenopus retains centrum primordia at the base of neural arches.

Intestinal remodeling during Xenopus metamorphosis as a model for studying thyroid hormone signaling and adult organogenesis.

Intestinal development takes places in two phases, the initial formation of neonatal (mammals)/larval (anurans) intestine and its subsequent maturation into the adult form. This maturation occurs during postembryonic development when plasma thyroid hormone (T3) level peaks. In anurans such as the highly related Xenopus laevis and Xenopus tropicalis, the larval/tadpole intestine is drastically remodeled from a simple tubular structure to a complex, multi-folded adult organ during T3-dependent metamorphosis. This involved complete degeneration of larval epithelium via programmed cell death and de novo formation of adult epithelium, with concurrent maturation of the muscles and connective tissue. Here, we will summarize our current understanding of the underlying molecular mechanisms, with a focus on more recent genetic and genome-wide studies.

Anatomical changes of Tenebrio molitor and Tribolium castaneum during complete metamorphosis.

In holometabolous insects, extensive reorganisation of tissues and cells occurs at the pupal stage. The remodelling of the external exoskeleton and internal organs that intervenes during metamorphosis has been traditionally studied in many insect species based on histological or ultrastructural methods. This study demonstrates the use of synchrotron X-ray phase-contrast micro-computed tomography as a powerful, non-destructive tool for in situ morphological observation of anatomical structures at the pupal stage in two Tenebrionid beetles, i.e. Tribolium castaneum and Tenebrio molitor, known as important pests, as well as emerging and promising models in experimental biology. Virtual sections and three-dimensional reconstructions were performed on both males and females at early, intermediate, and late pupal stage. The dataset allowed us to observe the remodelling of the gut and nervous system as well as the shaping of the female and male reproductive system at different pupal ages in both mealworm and red flour beetles. Moreover, we observed that the timing and duration pattern of organ development varied between the species analysed, likely related to the species-specific adaptations of the pre-imaginal stages to environmental conditions, which ultimately affect their life cycle. This research provides new knowledge on the morphological modifications that occur during the pupal stage of holometabolous insects and provides a baseline set of information on beetle metamorphosis that may support future research in forensics, physiology, and ecology as well as an image atlas for educational purposes.

Assessment of toxicity, genotoxicity and oxidative stress in Fejervarya limnocharis exposed to tributyltin.

Tributyltin (TBT) is widely used in various commercial applications due to its biocidal properties. Toxicological and genotoxicological data on TBT exposure to amphibians is insufficient. Our study aimed to determine the acute toxicity and genotoxic potential of TBT in Fejervarya limnocharis tadpoles. Furthermore, oxidative stress was also investigated in TBT-treated tadpoles. Tadpoles of Gosner stage (26-30) were screened and subjected to increasing concentrations of TBT (0, 3, 7, 11, 15, 19, 23 µg/L) for determining the LC50 values for 24 h, 48 h, 72 h, and 96 h. LC50 values of TBT for 24 h, 48 h, 72 h, and 96 h were found to be 19.45, 15.07, 13.12, and 11.84 µg/L respectively. Based on the 96 h LC50 value (11.84 µg/L), tadpoles were exposed to different sub-lethal concentrations of TBT for the evaluation of its genotoxic potential and effects on oxidative balance. The role of TBT on survivability, growth, and time to metamorphosis was also assessed. TBT exposure significantly altered the life history traits measured, increased mortality, and delayed the time taken to metamorphosis. Results indicated significant induction of micronucleus (MN, p < 0.001) and other erythrocytic nuclear aberrations (ENA, p < 0.01) in the TBT-treated groups. Significant alterations in comet parameters and oxidative balance were also observed in the treated groups. The present study findings might add to the cause of the gradual population decline seen in the amphibians. This study also demonstrates the alteration of the life-history traits, oxidative balance, and DNA damage upon TBT exposure which can have long-term consequences for the anuran amphibian F. limnocharis.

Chytridiomycosis causes high amphibian mortality prior to the completion of metamorphosis.

Amphibian populations are undergoing extensive declines globally. The fungal disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), is a primary contributor to these declines. The amphibian metamorphic stages (Gosner stages 42-46) are particularly vulnerable to a range of stressors, including Bd. Despite this, studies that explicitly examine host response to chytridiomycosis throughout the metamorphic stages are lacking. We aimed to determine how Bd exposure during the larval stages impacts metamorphic development and infection progression in the endangered Fleay's barred frog (Mixophyes fleayi). We exposed M. fleayi to Bd during pro-metamorphosis (Gosner stages 35-38) and monitored infection dynamics throughout metamorphosis. We took weekly morphological measurements (weight, total body length, snout-vent-length and Gosner stage) and quantified Bd load using qPCR. While we observed minimal impact of Bd infection on animal growth and development, Bd load varied throughout ontogeny, with an infection load plateau during the tadpole stages (Gosner stages 35-41) and temporary infection clearance at Gosner stage 42. Bd load increased exponentially between Gosner stages 42 and 45, with most exposed animals becoming moribund at Gosner stage 45, prior to the completion of metamorphosis. There was variability in infection outcome of exposed individuals, with a subgroup of animals (n = 5/29) apparently clearing their infection while the majority (n = 21/29) became moribund with high infection burdens. This study demonstrates the role that metamorphic restructuring plays in shaping Bd infection dynamics and raises the concern that substantial Bd-associated mortality could be overlooked in the field due to the often cryptic nature of these latter metamorphic stages. We recommend future studies that directly examine the host immune response to Bd infection throughout metamorphosis, incorporating histological and molecular methods to elucidate the mechanisms responsible for the observed trends.

Ecdysone and gene expressions for chromatin remodeling, histone modification, and Broad Complex in relation to pupal commitment in Bombyx mori.

In the present study, we tried to clarify when and how pupal commitment (PT) better to use PC occurs and what is involved in the PT of Bombyx mori. To clarify this, we examined the responsiveness of a wing disc to ecdysone, referring to metamorphosis-related BR-C, development-related Myc and Wnt, and chromatin remodeling-related genes at around the predicted PT stage of the Bombyx wing disc. Wing disc responsiveness to juvenile hormone (JH) and ecdysone was examined using Methoprene and 20-hydroxyecdysone (20E) in vitro. The body weight of B. mori increased after the last larval ecdysis, peaked at Day 5 of the fifth larval instar (D5L5), and then decreased. The responsiveness of the wing disc to JH decreased after the last larval ecdysis up to D3L5. Bmbr-c (the Broad Complex of B. mori) showed enhanced expression in D4L5 wing discs with 20E treatment. Some chromatin remodeler and histone modifier genes (Bmsnr1, Bmutx, and Bmtip60) showed upregulation after being cultured with 20E in D4L5 wing discs. A low concentration of 20E is suggested to induce responsiveness to 20E in D4L5 wing discs. Bmbr-c, Bmsnr1, Bmutx, and Bmtip60 were upregulated after being cultured with a low concentration of 20E in D4L5 wing discs. The expression of Bmmyc and Bmwnt1 did not show a change after being cultured with or without 20E in D4L5 wing discs, while enhanced expression was observed with 20E in D5L5 wing discs. From the present results, we concluded that PT of the wing disc of B. mori occurred beginning on D4L5 with the secretion of low concentrations of ecdysteroids. Bmsnr1, Bmutx, Bmtip60, and BR-C are also involved.

Wing expansion functional analysis of ion transport peptide gene in Sogatella furcifera (Horváth) (Hemiptera: Delphacidae).

Ion transport peptide (ITP), a superfamily of arthropod neuropeptides, serves a crucial role in regulating various physiological processes such as diuresis, ecdysis behavior, and wing expansion. However, the molecular characteristics, expression profile, and role of ITP in Sogatella furcifera are poorly understood. To elucidate the characteristics and biological function of ITP in S. furcifera, we employed reverse transcription-polymerase chain reaction (RT-PCR) and RNA interference (RNAi) methods. The identified SfITP gene encodes 117 amino acids. The expression of SfITP gradually increased followed the formation of 3-day-old of 5th instar nymph, peaking initially at 40 min after eclosion, and reaching another peak 24 h after eclosion, with particularly high expression levels in thorax and wing tissues. Notably, SfITP RNAi in 3rd instar nymphs of S. furcifera significantly inhibited the transcript levels of SfITP, resulting in 55% mortality and 78% wing deformity. These findings suggests that SfITP is involved in the regulation of wing expansion in S. furcifera, providing insights into the regulation of insect wing expansion and contributing to the molecular understanding of this process.

Carbon nitride dots do not impair the growth, development, and telomere length of tadpoles.

Carbon nanoparticles, or carbon dots, can have many beneficial uses. However, we must consider whether they may have any potential negative side effects on wildlife or the ecosystem when these particles end up in wastewater. Early development stages of amphibians are particularly sensitive to contaminants, and exposure to carbon dots could disrupt their development and cause morbidity or death. Past studies have investigated short-term exposure to certain types of nanoparticles, but if these particles get into wastewater exposure may not be short term. Therefore, we tested whether chronic exposure to different concentrations of carbon dots affects the growth, metamorphosis, and telomere length of Cuban tree frog (Osteopilus septentrionalis) tadpoles. We exposed 12 groups of five tadpoles each to different concentrations of carbon dots and a control for three months and tracked survival, growth and metamorphosis. We used carbon nitride dots approximately 2 nm in size at concentrations of 0.01 mg/ml and 0.02 mg/ml, known to interrupt development in zebrafish embryos. After three months, we measured telomere length from tissue samples. We found no difference in tadpole survivorship, growth, development rate, or telomere length among any of the groups, suggesting that carbon dots at these concentrations do not disrupt tadpole development.

Utilization of temperature-mediated activation of thyroid hormone-induced molecular memory to evaluate early signaling events in the olfactory epithelium of Rana [Lithobates] catesbeiana tadpoles.

The amphibian olfactory system is highly distinct between aquatic tadpole and terrestrial frog life stages and therefore must remodel extensively during thyroid hormone (TH)-dependent metamorphosis. Developmentally appropriate functioning of the olfactory epithelium is critical for survival. Previous studies in other Rana [Lithobates] catesbeiana premetamorphic tadpole tissues showed that initiation of TH-induced metamorphosis can be uncoupled from execution of TH-dependent programs by holding tadpoles in the cold rather than at warmer permissive temperatures. TH-exposed tadpoles at the nonpermissive (5 °C) temperature do not undergo metamorphosis but retain a "molecular memory" of TH exposure that is activated upon shift to a permissive warm temperature. Herein, premetamorphic tadpoles were held at permissive (24 °C) or nonpermissive (5 °C) temperatures and injected with 10 pmoles/g body weight 3,5,3'-triiodothyronine (T3) or solvent control. Olfactory epithelium was collected at 48 h post-injection. RNA-sequencing (RNA-Seq) and reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analyses generated differentially expressed transcript profiles of 4328 and 54 contigs for permissive and nonpermissive temperatures, respectively. Translation, rRNA, spliceosome, and proteolytic processes gene ontologies were enriched by T3 treatment at 24 °C while negative regulation of cell proliferation was enriched by T3 at 5 °C. Of note, as found in other tissues, TH-induced basic leucine zipper-containing protein-encoding transcript, thibz, was significantly induced by T3 at both temperatures, suggesting a role in the establishment of molecular memory in the olfactory epithelium. The current study provides critical insights by deconstructing early TH-induced induction of postembryonic processes that may be targets for disruption by environmental contaminants.

Ontogeny of the Cytochrome P450 Superfamily in the Ornate Spiny Lobster (Panulirus ornatus).

Cytochrome P450s (CYP450s) are a versatile superfamily of enzymes known to undergo rapid evolution. They have important roles across growth and development pathways in crustaceans, although it is difficult to characterise orthologs between species due to their sequence diversity. Conserved CYP450s enzymes in crustaceans are those associated with ecdysteroidogenesis: synthesising and breaking down the active moult hormone, 20-hydroxyecdysone. The complex life cycle of the ornate spiny lobster, Panulirus ornatus, relies on moulting in order to grow and develop. Many of these diverse life stages have been analysed to establish a comprehensive transcriptomic database for this species. The transcripts putatively encoding for CYP450s were mapped using transcriptomic analysis and identified across growth and development stages. With the aid of phylogeny, 28 transcripts of 42 putative P. ornatus CYP450s were annotated, including the well conserved Halloween genes, which are involved in ecdysteroidogenesis. Expression patterns across the life stages determined that only a subset of the CYP450s can be detected in each life stage or tissue. Four Shed transcripts show overlapping expression between metamorphosis and adult tissues, suggesting pleotropic functions of the multiple Shed orthologs within P. ornatus.