Intraspecific priority effects in response to egg hatching delay in a pond-breeding salamander.

As reproduction phenologies shift with climate change, populations can experience intraspecific priority effects, wherein early hatching cohorts experience an advantage over late-hatching cohorts, resulting in altered demography. Our study objective was to identify how variation in egg hatching phenology alters intraspecific interactions in small-mouthed salamanders, Ambystoma texanum. We addressed two research questions: (Q1) How are demographic responses altered by variation in the temporal duration of hatching between cohorts, and (Q2) How does the seasonality of hatching delays affect demographic responses? We manipulated hatching phenologies of A. texanum eggs and reared larvae in outdoor mesocosms to metamorphosis. For Q1, hatching delay exhibited non-linear relationships with survival and body size, with the greatest asynchrony in cohort additions resulting in the highest mortality and largest body sizes. For Q2, hatching delay effects were stronger (i.e., survival was lower and body sizes larger) when they occurred later in the season, potentially due to temperature differences that larvae experienced. Overall, our results demonstrate that changes in intraspecific interactions due to phenological shifts can be context-dependent, depending on the strength (i.e., temporal duration) and seasonality of such processes. Identifying context-dependencies of phenological shifts will be critical for predicting changes in organismal demographics with climatic shifts.

The transcriptional landscape underlying larval development and metamorphosis in the Malabar grouper (Epinephelus malabaricus).

Most teleost fishes exhibit a biphasic life history with a larval oceanic phase that is transformed into morphologically and physiologically different demersal, benthic, or pelagic juveniles. This process of transformation is characterized by a myriad of hormone-induced changes, during the often abrupt transition between larval and juvenile phases called metamorphosis. Thyroid hormones (TH) are known to be instrumental in triggering and coordinating this transformation but other hormonal systems such as corticoids, might be also involved as it is the case in amphibians. In order to investigate the potential involvement of these two hormonal pathways in marine fish post-embryonic development, we used the Malabar grouper (Epinephelus malabaricus) as a model system. We assembled a chromosome-scale genome sequence and conducted a transcriptomic analysis of nine larval developmental stages. We studied the expression patterns of genes involved in TH and corticoid pathways, as well as four biological processes known to be regulated by TH in other teleost species: ossification, pigmentation, visual perception, and metabolism. Surprisingly, we observed an activation of many of the same pathways involved in metamorphosis also at an early stage of the larval development, suggesting an additional implication of these pathways in the formation of early larval features. Overall, our data brings new evidence to the controversial interplay between corticoids and thyroid hormones during metamorphosis as well as, surprisingly, during the early larval development. Further experiments will be needed to investigate the precise role of both pathways during these two distinct periods and whether an early activation of both corticoid and TH pathways occurs in other teleost species.

Molecular basis of urostyle development in frogs: genes and gene regulation underlying an evolutionary novelty.

Evolutionary novelties entail the origin of morphologies that enable new functions. These features can arise through changes to gene function and regulation. One key novelty is the fused rod at the end of the vertebral column in anurans, the urostyle. This feature is composed of a coccyx and a hypochord, both of which ossify during metamorphosis. To elucidate the genetic basis of these features, we used laser capture microdissection of these tissues and did RNA-seq and ATAC-seq at three developmental stages in tadpoles of Xenopus tropicalis. RNA-seq reveals that the coccyx and hypochord have two different molecular signatures. Neuronal (TUBB3) and muscle markers (MYH3) are upregulated in coccygeal tissues, whereas T-box genes (TBXT, TBXT.2), corticosteroid stress hormones (CRCH.1) and matrix metallopeptidases (MMP1, MMP8 and MMP13) are upregulated in the hypochord. ATAC-seq reveals potential regulatory regions that are observed in proximity to candidate genes that regulate ossification identified from RNA-seq. Even though an ossifying hypochord is only present in anurans, this ossification between the vertebral column and the notochord resembles a congenital vertebral anomaly seen prenatally in humans caused by an ectopic expression of the TBXT/TBXT.2 gene. This work opens the way to functional studies that can elucidate anuran bauplan evolution.

The impact of chemical pollution across major life transitions: a meta-analysis on oxidative stress in amphibians.

Among human actions threatening biodiversity, the release of anthropogenic chemical pollutants which have become ubiquitous in the environment, is a major concern. Chemical pollution can induce damage to macromolecules by causing the overproduction of reactive oxygen species, affecting the redox balance of animals. In species undergoing metamorphosis (i.e. the vast majority of the extant animal species), antioxidant responses to chemical pollution may differ between pre- and post-metamorphic stages. Here, we meta-analysed (N = 104 studies, k = 2283 estimates) the impact of chemical pollution on redox balance across the three major amphibian life stages (embryo, tadpole, adult). Before metamorphosis, embryos did not experience any redox change while tadpoles activate their antioxidant pathways and do not show increased oxidative damage from pollutants. Tadpoles may have evolved stronger defences against pollutants to reach post-metamorphic life stages. In contrast, post-metamorphic individuals show only weak antioxidant responses and marked oxidative damage in lipids. The type of pollutant (i.e. organic versus inorganic) has contrasting effects across amphibian life stages. Our findings show a divergent evolution of the redox balance in response to pollutants across life transitions of metamorphosing amphibians, most probably a consequence of differences in the ecological and developmental processes of each life stage.

Developmental temperature modulates microplastics impact on amphibian life history without affecting ontogenetic microplastic transfer.

This study examines how temperature influences the response of Japanese tree frogs (Dryophytes japonicus) to microplastic (MP) pollution, assessing whether temperature can regulate the harmful effects of MPs on their life history and the dispersal of MPs across habitats. This analysis aims to understand the ecological and physiological ramifications of MP pollution. Our results demonstrated an ontogenetic transfer of MP particles across amphibian metamorphosis, possibly allowing and facilitating the translocation of MPs across ecosystems. Temperature did not significantly affect the translocation of aquatic MPs to land. However, high temperatures significantly reduced mortality and hindlimb deformities caused by MPs, thereby mitigating their harmful impact on amphibian life histories. Importantly, our study found that MPs cause hindlimb deformities during amphibian metamorphosis, potentially linked to oxidative stress. Additionally, MP exposure and ingestion induced a plastic response in the morphology of the digestive tract and changes in the fecal microbiome, which were evident at high temperatures but not at low temperatures. The effects of MPs persisted even after the frogs transitioned to the terrestrial stage, suggesting that MPs may have complex, long-term impacts on amphibian population sustainability. Our results enhance the understanding of the intricate environmental challenges posed by MPs and underscore the significant role of temperature in ectotherms regarding ontogenetic impacts and pollutant interactions.

Lipases are differentially regulated by hormones to maintain free fatty acid homeostasis for insect brain development.

BACKGROUND: Free fatty acids (FFAs) play vital roles as energy sources and substrates in organisms; however, the molecular mechanism regulating the homeostasis of FFA levels in various circumstances, such as feeding and nonfeeding stages, is not fully clarified. Holometabolous insects digest dietary triglycerides (TAGs) during larval feeding stages and degrade stored TAGs in the fat body during metamorphosis after feeding cessation, which presents a suitable model for this study. RESULTS: This study reported that two lipases are differentially regulated by hormones to maintain the homeostasis of FFA levels during the feeding and nonfeeding stages using the lepidopteran insect cotton bollworm Helicoverpa armigera as a model. Lipase member H-A-like (Lha-like), related to human pancreatic lipase (PTL), was abundantly expressed in the midgut during the feeding stage, while the monoacylglycerol lipase ABHD12-like (Abhd12-like), related to human monoacylglycerol lipase (MGL), was abundantly expressed in the fat body during the nonfeeding stage. Lha-like was upregulated by juvenile hormone (JH) via the JH intracellular receptor methoprene-tolerant 1 (MET1), and Abhd12-like was upregulated by 20-hydroxyecdysone (20E) via forkhead box O (FOXO) transcription factor. Knockdown of Lha-like decreased FFA levels in the hemolymph and reduced TAG levels in the fat body. Moreover, lipid droplets (LDs) were small, the brain morphology was abnormal, the size of the brain was small, and the larvae showed the phenotype of delayed pupation, small pupae, and delayed tissue remodeling. Knockdown of Abhd12-like decreased FFA levels in the hemolymph; however, TAG levels increased in the fat body, and LDs remained large. The development of the brain was arrested at the larval stage, and the larvae showed a delayed pupation phenotype and delayed tissue remodeling. CONCLUSIONS: The differential regulation of lipases expression by different hormones determines FFAs homeostasis and different TAG levels in the fat body during the feeding larval growth and nonfeeding stages of metamorphosis in the insect. The homeostasis of FFAs supports insect growth, brain development, and metamorphosis.

Insect cuticular protein; gene expression, genomic structure, transcriptional regulation, speculated cuticular structure, clarified through the genomic analysis of Bombyx mori.

JH and ecdysone signaling regulate insect metamorphosis through the master transcription factors, Krüppel homolog 1 (kr-h1), Broad-Complex (BR-C), and E93. Ecdysone signaling activates successively expressed ecdysone responsive transcription factors (ERTFs), and the interaction between ERTFs determines the expression profiles of ERTFs themselves. Through the construction of expressed sequence tag (EST) database of Bombyx mori from many tissues, the existence of a large number of cuticular protein (CP) genes was identified in wing disc cDNA library of the 3 days after the start of wandering (W3). From the genomic analysis, 12 types of CP clusters of CP genes were identified. DNA sequences of CP genes revealed the duplication of CP genes, which suggests to reflect the insect evolution. These CP genes responded to ecdysone and ecdysone pulse; therefore, CP genes were applied for the analysis of transcriptional regulation by ERTF. The binding sites of ERTF have been reported to exist upstream of CP genes in several insects, and the activation of CP genes occurred by the binding of ERTFs. Through the analysis, the following were speculated; the successive appearance of ERTFs and the activation of target genes resulted in the successively produced CPs and cuticular layer. The sequence of the ERTF and CP gene expression was the same at larval to pupal and pupal to adult transformation. The involvement of several ERTFs in one CP gene expression was also clarified; BmorCPG12 belongs to group showing expression peak at W3 and was regulated by two ERTFs; BHR3 and ßFTZ-F1, BmorCPH2 belongs to group showing expression peak at P0 and was regulated by two ERTFs; ßFTZ-F1 and E74A. The involvement of BHR39 as a negative regulator of CP gene expression was found. Larval, pupal, and adult cuticular layers were supposed to be constructed by the combination of different and similar types of CPs, through the expressed timing of CP genes.

A comprehensive molecular phylogeny of the terrestrial Parasitengona (Acariformes, Prostigmata) provides insights into the evolution of their metamorphosis, invasion into aquatic habitats and classification.

Parasitengona (velvet mites, chiggers and water mites) is a highly diverse and globally distributed mite lineage encompassing over 11,000 described species, inhabiting terrestrial, freshwater and marine habitats. Certain species, such as chiggers (Trombiculidae), have a great medical and veterinary importance as they feed on their vertebrate hosts and vector pathogens. Despite extensive previous research, the classification of Parasitengona is still contentious, particularly regarding the boundaries between superfamilies and families, exacerbated by the absence of a comprehensive phylogeny. The ontogeny of most Parasitengona is distinct by the presence of striking metamorphosis, with parasitic larvae being heteromorphic compared to the predatory free-living deutonymphs and adults. The enigmatic superfamily Allotanaupodoidea is an exception, with larvae and active post-larval stages being morphologically similar, suggesting that the absence of metamorphosis may be either an ancestral state or a secondary reversal. Furthermore, there is disagreement in the literature on whether Parasitengona had freshwater or terrestrial origin. Here, we inferred phylogenetic relationships of Parasitengona (89 species, 36 families) and 307 outgroups using five genes (7,838 nt aligned). This phylogeny suggests a terrestrial origin of Parasitengona and a secondary loss of metamorphosis in Allotanaoupodoidea. We recovered the superfamily Trombidioidea (Trombidioidea sensu lato) as a large, well-supported, higher-level clade including 10 sampled families. We propose a new classification for the terrestrial Parasitengona with three new major divisions (epifamilies) of the superfamily Trombidioidea: Trombelloidae (families Audyanidae, Trombellidae, Neotrombidiidae, Johnstonianidae, Chyzeriidae); Trombidioidae (Microtrombidiidae, Neothrombiidae, Achaemenothrombiidae, Trombidiidae, Podothrombiidae); and Trombiculoidae (=Trombiculidae sensu lato). Adding them to previously recognized superfamilies Allotanaupodoidea, Amphotrombioidea, Calyptostomatoidea, Erythraeoidea, Tanaupodoidae and Yurebilloidae.

Longer days, larger grays: carryover effects of photoperiod and temperature in gray treefrogs, Hyla versicolor.

Environmental conditions like temperature and photoperiod can strongly shape organisms' growth and development. For many ectotherms with complex life cycles, global change will cause their offspring to experience warmer conditions and earlier-season photoperiods, two variables that can induce conflicting responses. We experimentally manipulated photoperiod and temperature during gray treefrog (Hyla versicolor) larval development to examine effects at metamorphosis and during short (10-day) and long (56-day) periods post-metamorphosis. Both early- and late-season photoperiods (April and August) decreased age and size at metamorphosis relative to the average-season (June) photoperiod, while warmer temperatures decreased age but increased size at metamorphosis. Warmer larval temperatures reduced short-term juvenile growth but had no long-term effect. Conversely, photoperiod had no short-term carryover effect, but juveniles from early- and late-season larval photoperiods had lower long-term growth rates than juveniles from the average-season photoperiod. Similar responses to early- and late-season photoperiods may be due to reduced total daylight compared with average-season photoperiods. However, juveniles from late-season photoperiods selected cooler temperatures than early-season juveniles, suggesting that not all effects of photoperiod were due to total light exposure. Our results indicate that despite both temperature and photoperiod affecting metamorphosis, the long-term effects of photoperiod may be much stronger than those of temperature.

Effects of warming at embryonic and larval stages on tadpole fitness in high-altitude Rana kukunoris.

Global warming may affect the early developmental stages of high-altitude amphibians, thereby influencing their later fitness. Yet, this has been largely unexplored. To investigate whether and how the temperatures experienced by embryonic and larval stages affect their fitness at later developmental stages, we designed two experiments in which the embryos and larvae were treated with three temperatures (24, 18 and 12 °C), respectively. Then, the life history traits of the tadpoles during the metamorphotic climax in all treatments were evaluated, including growth rate, survival rate, morphology, thermal physiology, swimming performance, standard metabolic rate (SMR), oxidative and antioxidative system, and metabolic enzyme activities. The results revealed that elevated temperature accelerated metamorphosis but decreased body size at metamorphosis. Additionally, warming during the embryonic and larval stages decreased the thermal tolerance range and induced increased oxidative stress. Furthermore, high embryonic temperature significantly decreased the hatching success, but had no significant effect on swimming performance and SMR. Warming during larval periods was harmful to the survival and swimming performance of tadpoles. The effect size analysis revealed that the negative impacts of embryonic temperature on certain physiological traits, such as growth and development, survival and swimming performance, were more pronounced than those of larval temperature. Our results highlight the necessity for particular attention to be paid to the early stages of amphibians, notably the embryonic stages when evaluating the impact of global warming on their survival.

Integrated biomarker-based ecological risks assessment of tadpole responses to tris(2-chloroethyl) phosphate, tris(1-chloro-2-propyl) phosphate, and their combined environmental exposure.

Tris(2-chloroethyl) phosphate (TCEP) and tris(1-chloro-2-propyl) phosphate (TCPP) are common chlorinated organophosphorus flame retardants (OPFRs) used in industry. They have been frequently detected together in aquatic environments and associated with various hazardous effects. However, the ecological risks of prolonged exposure to these OPFRs at environmentally relevant concentrations in non-model aquatic organisms remain unexplored. This study investigated the effects of long-term exposure (up to 25 days) to TCEP and TCPP on metamorphosis, hepatic antioxidants, and endocrine function in Polypedates megacephalus tadpoles. Exposure concentrations were set at 3, 30, and 90 µg/L for each substance, conducted independently and in equal-concentration combinations, with a control group included for comparison. The integrated biomarker response (IBR) method developed an optimal linear model for predicting the overall ecological risks of TCEP and TCPP to tadpoles in potential distribution areas of Polypedates species. Results showed that: (1) Exposure to environmentally relevant concentrations of TCEP and TCPP elicited variable adverse effects on tadpole metamorphosis time, hepatic antioxidant enzyme activity and related gene expression, and endocrine-related gene expression, with their combined exposure exacerbating these effects. (2) The IBR value of TCEP was consistently greater than that of TCPP at each concentration, with an additive effect observed under their combined exposure. (3) The ecological risk of tadpoles exposed to the combined presence of TCEP and TCPP was highest in China's Taihu Lake and Vietnam's Hanoi than in other distribution locations. In summary, prolonged exposure to environmentally relevant concentrations of TCEP and TCPP presents potential ecological risks to amphibian tadpoles, offering insights for the development of policies and strategies to control TCEP and TCPP pollution in aquatic ecosystems. Furthermore, the methodology employed in establishing the IBR prediction model provides a methodological framework for assessing the overall ecological risks of multiple OPFRs.

Dynamic Alternative Polyadenylation during Litopenaeus Vannamei Metamorphosis Development.

As an important mechanism in the post-transcriptional regulation of eukaryotic gene expression, alternative polyadenylation (APA) plays a key role in biological processes such as cell proliferation and differentiation. However, the role and dynamic pattern of APA during Litopenaeus vannamei metamorphosis are poorly understood. Here, RNA-seq data covering from the embryo to the maturation (16 time points) of L. vannamei were utilized. We identified 247 differentially expressed APA events between early and adult stages, and through fuzzy mean clustering analysis, we discovered five dynamic APA patterns. Among them, the gradual elongation of the 3'UTR is the major APA pattern that changes over time, and its genes are enriched in the pathways of protein and energy metabolism. Finally, we constructed mRNA-miRNA and PPI networks and detected several central miRNAs that may regulate L. vannamei development. Our results revealed the complex APA mechanisms in L. vannamei metamorphosis, shedding new light on post-transcriptional regulation of crustacean metamorphosis.

Bacterial envelope polysaccharide cues settlement and metamorphosis in the biofouling tubeworm Hydroides elegans.

Metamorphosis for many marine invertebrates is triggered by external cues, commonly produced by bacteria. For larvae of Hydroides elegans, lipopolysaccharide (LPS) from the biofilm-dwelling bacterium Cellulophaga lytica induces metamorphosis. To determine whether bacterial LPS is a common metamorphosis-inducing factor for this species, we compare larval responses to LPS from 3 additional inductive Gram-negative marine biofilm bacteria with commercially available LPS from 3 bacteria not known to induce metamorphosis. LPS from all the inductive bacteria trigger metamorphosis, while LPS from non-inductive isolated marine bacteria do not. We then ask, which part of the LPS is the inductive element, the lipid (Lipid-A) or the polysaccharide (O-antigen), and find it is the latter for all four inductive bacteria. Finally, we examine the LPS subunits from two strains of the same bacterial species, one inductive and the other not, and find the LPS and O-antigen to be inductive from only the inductive bacterial strain.

Unveiling vertebrate development dynamics in frog Xenopus laevis using micro-CT imaging.

BACKGROUND: Xenopus laevis, the African clawed frog, is a versatile vertebrate model organism in various biological disciplines, prominently in developmental biology to study body plan reorganization during metamorphosis. However, a notable gap exists in the availability of comprehensive datasets encompassing Xenopus' late developmental stages. FINDINGS: This study utilized micro-computed tomography (micro-CT), a noninvasive 3-dimensional (3D) imaging technique with micrometer-scale resolution, to explore the developmental dynamics and morphological changes in Xenopus laevis. Our approach involved generating high-resolution images and computed 3D models of developing Xenopus specimens, spanning from premetamorphosis tadpoles to fully mature adults. This dataset enhances our understanding of vertebrate development and supports various analyses. We conducted a careful examination, analyzing body size, shape, and morphological features, focusing on skeletogenesis, teeth, and organs like the brain and gut at different stages. Our analysis yielded valuable insights into 3D morphological changes during Xenopus' development, documenting details previously unrecorded. These datasets hold the solid potential for further morphological and morphometric analyses, including segmentation of hard and soft tissues. CONCLUSIONS: Our repository of micro-CT scans represents a significant resource that can enhance our understanding of Xenopus' development and the associated morphological changes in the future. The widespread utility of this amphibian species, coupled with the exceptional quality of our scans, which encompass a comprehensive series of developmental stages, opens up extensive opportunities for their broader research application. Moreover, these scans can be used in virtual reality, 3D printing, and educational contexts, further expanding their value and impact.

The metamorphosis of amphibian myocardium: moving to the heart of the matter.

Amphibians are a classical object for physiological studies, and they are of great value for developmental studies owing to their transition from an aquatic larval form to an adult form with a terrestrial lifestyle. Axolotls (Ambystoma mexicanum) are of special interest for such studies because of their neoteny and facultative pedomorphosis, as in these animals, metamorphosis can be induced and fully controlled in laboratory conditions. It has been suggested that their metamorphosis, associated with gross anatomical changes in the heart, also involves physiological and electrical remodeling of the myocardium. We used whole-cell patch clamp to investigate possible changes caused by metamorphosis in electrical activity and major ionic currents in cardiomyocytes isolated from paedomorphic and metamorphic axolotls. T4-induced metamorphosis caused shortening of atrial and ventricular action potentials (APs), with no changes in resting membrane potential or maximum velocity of AP upstroke, favoring higher heart rate possible in metamorphic animals. Potential-dependent potassium currents in axolotl myocardium were represented by delayed rectifier currents IKr and IKs, and upregulation of IKs caused by metamorphosis probably underlies AP shortening. Metamorphosis was associated with downregulation of inward rectifier current IK1, probably serving to increase the excitability of myocardium in metamorphic animals. Metamorphosis also led to a slight increase in fast sodium current INa with no changes in its steady-state kinetics and to a significant upregulation of ICa in both atrial and ventricular cells, indicating stronger Ca2+ influx for higher cardiac contractility in metamorphic salamanders. Taken together, these changes serve to increase cardiac reserve in metamorphic animals.

Cadmium bioaccumulation dynamics during amphibian development and metamorphosis.

Cadmium pollution poses a significant threat to aquatic ecosystems due to its propensity to bioaccumulate and cause toxicity. This study assessed the complex dynamics of cadmium uptake, accumulation and distribution across anuran development to provide new insights into the fate of cadmium burdens during metamorphosis and compare the susceptibility of different life stages to cadmium accumulation. Tadpoles of various developmental stages were exposed to dissolved 109-cadmium and depurated in clean water in a series of experiments. Temporal changes in whole-body and tissue concentrations were analysed using gamma spectroscopy, and anatomical distributions were visualised using autoradiography. Results showed that animals exposed at the onset of metamorphic climax (forelimb emergence) retained significantly less cadmium than animals exposed through larval stages. After exposure, cadmium partitioned predominantly in the skin, gills and remains of metamorphs, whereas larvae accumulated cadmium predominately through their gut. This shows a shift in the primary route of uptake at the onset of climax, which relates to the structural and functional changes of uptake sites through metamorphosis. During climax, some cadmium was redistributed in tissues developing de novo, such as the forelimbs, and concentrated in the regressing tail. Our findings highlight the need for stage-specific considerations in assessing exposure risks.

Isoforms of terminal selector Mamo control axon guidance during adult Drosophila memory center construction via Semaphorin-1a.

In animals undergoing metamorphosis, the appearance of the nervous system is coincidently transformed by the morphogenesis of neurons. Such morphogenic alterations are exemplified in three types of intrinsic neurons in the Drosophila memory center. In contrast to the well-characterized remodeling of γ neurons, the morphogenesis of α/ß and α'/ß' neurons has not been adequately explored. Here, we show that mamo, a BTB-zinc finger transcription factor that acts as a terminal selector for α'/ß' neurons, controls the formation of the correct axonal pattern of α'/ß' neurons. Intriguingly, specific Mamo isoforms are preferentially expressed in α'/ß' neurons to regulate the expression of axon guidance molecule Semaphorin-1a. This action directs proper axon guidance in α'/ß' neurons, which is also crucial for wiring of α'/ß' neurons with downstream neurons. Taken together, our results provide molecular insights into how neurons establish correct axonal patterns in circuitry assembly during adult memory center construction.

Transcriptome analysis of the Japanese eel (Anguilla japonica) during larval metamorphosis.

BACKGROUND: Anguillid eels spend their larval period as leptocephalus larvae that have a unique and specialized body form with leaf-like and transparent features, and they undergo drastic metamorphosis to juvenile glass eels. Less is known about the transition of leptocephali to the glass eel stage, because it is difficult to catch the metamorphosing larvae in the open ocean. However, recent advances in rearing techniques for the Japanese eel have made it possible to study the larval metamorphosis of anguillid eels. In the present study, we investigated the dynamics of gene expression during the metamorphosis of Japanese eel leptocephali using RNA sequencing. RESULTS: During metamorphosis, Japanese eels were classified into 7 developmental stages according to their morphological characteristics, and RNA sequencing was used to collect gene expression data from each stage. A total of 354.8 million clean reads were generated from the body and 365.5 million from the head, after the processing of raw reads. For filtering of genes that characterize developmental stages, a classification model created by a Random Forest algorithm was built. Using the importance of explanatory variables feature obtained from the created model, we identified 46 genes selected in the body and 169 genes selected in the head that were defined as the "most characteristic genes" during eel metamorphosis. Next, network analysis and subsequently gene clustering were conducted using the most characteristic genes and their correlated genes, and then 6 clusters in the body and 5 clusters in the head were constructed. Then, the characteristics of the clusters were revealed by Gene Ontology (GO) enrichment analysis. The expression patterns and GO terms of each stage were consistent with previous observations and experiments during the larval metamorphosis of the Japanese eel. CONCLUSION: Genome and transcriptome resources have been generated for metamorphosing Japanese eels. Genes that characterized metamorphosis of the Japanese eel were identified through statistical modeling by a Random Forest algorithm. The functions of these genes were consistent with previous observations and experiments during the metamorphosis of anguillid eels.

Tadpoles rely on mechanosensory stimuli for communication when visual capabilities are poor.

The ways in which animals sense the world changes throughout development. For example, young of many species have limited visual capabilities, but still make social decisions, likely based on information gathered through other sensory modalities. Poison frog tadpoles display complex social behaviors that have been suggested to rely on vision despite a century of research indicating tadpoles have poorly-developed visual systems relative to adults. Alternatively, other sensory modalities, such as the lateral line system, are functional at hatching in frogs and may guide social decisions while other sensory systems mature. Here, we examined development of the mechanosensory lateral line and visual systems in tadpoles of the mimic poison frog (Ranitomeya imitator) that use vibrational begging displays to stimulate egg feeding from their mothers. We found that tadpoles hatch with a fully developed lateral line system. While begging behavior increases with development, ablating the lateral line system inhibited begging in pre-metamorphic tadpoles, but not in metamorphic tadpoles. We also found that the increase in begging and decrease in reliance on the lateral line co-occurs with increased retinal neural activity and gene expression associated with eye development. Using the neural tracer neurobiotin, we found that axonal innervations from the eye to the brain proliferate during metamorphosis, with few retinotectal connections in recently-hatched tadpoles. We then tested visual function in a phototaxis assay and found tadpoles prefer darker environments. The strength of this preference increased with developmental stage, but eyes were not required for this behavior, possibly indicating a role for the pineal gland. Together, these data suggest that tadpoles rely on different sensory modalities for social interactions across development and that the development of sensory systems in socially complex poison frog tadpoles is similar to that of other frog species.

Localization and origins of juvenile skeletogenic cells in the sea urchin Lytechinuspictus.

The development of the sea urchin larval body plan is well understood from extensive studies of embryonic patterning. However, fewer studies have investigated the late larval stages during which the unique pentaradial adult body plan develops. Previous work on late larval development highlights major tissue changes leading up to metamorphosis, but the location of specific cell types during juvenile development is less understood. Here, we improve on technical limitations by applying highly sensitive hybridization chain reaction fluorescent in situ hybridization (HCR-FISH) to the fast-developing and transparent sea urchin Lytechinus pictus, with a focus on skeletogenic cells. First, we show that HCR-FISH can be used in L. pictus to precisely localize skeletogenic cells in the rudiment. In doing so, we provide a detailed staging scheme for the appearance of skeletogenic cells around the rudiment prior to and during biomineralization and show that many skeletogenic cells unassociated with larval rods localize outside of the rudiment prior to localizing inside. Second, we show that downstream biomineralization genes have similar expression patterns during larval and juvenile skeletogenesis, suggesting some conservation of skeletogenic mechanisms during development between stages. Third, we find co-expression of blastocoelar and skeletogenic cell markers around juvenile skeleton located outside of the rudiment, which is consistent with data showing that cells from the non-skeletogenic mesoderm embryonic lineage contribute to the juvenile skeletogenic cell lineage. This work sets the foundation for subsequent studies of other cell types in the late larva of L. pictus to better understand juvenile body plan development, patterning, and evolution.