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A complete larval zebrafish brain is examined and its myelinated axons reconstructed using serial-section electron microscopy, revealing remarkable symmetry and providing a valuable resource. Mapping the zebrafish brain Reconstructing neuronal circuits through serial-section electron microscopy (ssEM) requires a sub-nanoscale resolution that is more than 10 orders of magnitude smaller than whole vertebrate brains. This has limited connectomics efforts to elucidate restricted circuits. Florian Engert and colleagues report the ssEM reconstruction of a complete larval zebrafish brain, which reveals remarkable bilateral symmetry in the myelinated axon 'projectome'. The work further illustrates how such datasets can guide co-registering of structural and functional imaging data from a same specimen. High-resolution serial-section electron microscopy (ssEM) makes it possible to investigate the dense meshwork of axons, dendrites, and synapses that form neuronal circuits 1 . However, the imaging scale required to comprehensively reconstruct these structures is more than ten orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons 2 , some of which span nearly the entire brain. Difficulties in generating and handling data for large volumes at nanoscale resolution have thus restricted vertebrate studies to fragments of circuits. These efforts were recently transformed by advances in computing, sample handling, and imaging techniques 1 , but high-resolution examination of entire brains remains a challenge. Here, we present ssEM data for the complete brain of a larval zebrafish ( Danio rerio ) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to survey all myelinated axons (the projectome). These reconstructions enable precise investigations of neuronal morphology, which reveal remarkable bilateral symmetry in myelinated reticulospinal and lateral line afferent axons. We further set the stage for whole-brain structure–function comparisons by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. All obtained images and reconstructions are provided as an open-access resource.

Indirect development with an intermediate larva exists in all major animal lineages1, which makes larvae central to most scenarios of animal evolution2,3,4,5,6,7,8,9,10,11. Yet how larvae evolved remains disputed. Here we show that temporal shifts (that is, heterochronies) in trunk formation underpin the diversification of larvae and bilaterian life cycles. We performed chromosome-scale genome sequencing in the annelid Owenia fusiformis with transcriptomic and epigenomic profiling during the life cycles of this and two other annelids. We found that trunk development is deferred to pre-metamorphic stages in the feeding larva of O. fusiformis but starts after gastrulation in the non-feeding larva with gradual metamorphosis of Capitella teleta and the direct developing embryo of Dimorphilus gyrociliatus. Accordingly, the embryos of O. fusiformis develop first into an enlarged anterior domain that forms larval tissues and the adult head12. Notably, this also occurs in the so-called ‘head larvae’ of other bilaterians13,14,15,16,17, with which the O. fusiformis larva shows extensive transcriptomic similarities. Together, our findings suggest that the temporal decoupling of head and trunk formation, as maximally observed in head larvae, facilitated larval evolution in Bilateria. This diverges from prevailing scenarios that propose either co-option9,10 or innovation11 of gene regulatory programmes to explain larva and adult origins.

Modern biomedical research and preclinical pharmaceutical development rely heavily on the phenotyping of small vertebrate models for various diseases prior to human testing. In this article, we demonstrate an acoustofluidic rotational tweezing platform that enables contactless, high-speed, 3D multispectral imaging and digital reconstruction of zebrafish larvae for quantitative phenotypic analysis. The acoustic-induced polarized vortex streaming achieves contactless and rapid (~1 s/rotation) rotation of zebrafish larvae. This enables multispectral imaging of the zebrafish body and internal organs from different viewing perspectives. Moreover, we develop a 3D reconstruction pipeline that yields accurate 3D models based on the multi-view images for quantitative evaluation of basic morphological characteristics and advanced combinations of metrics. With its contactless nature and advantages in speed and automation, our acoustofluidic rotational tweezing system has the potential to be a valuable asset in numerous fields, especially for developmental biology, small molecule screening in biochemistry, and pre-clinical drug development in pharmacology. Existing methods of zebrafish phenotyping rely on contact-based processes. Here the authors report on an acoustofluidic-based platform which performs contactless specimen rotation, that results in multispectral images for rapid morphological phenotyping of zebrafish larvae.

Ciona robusta ( Ciona intestinalis type A), a model organism for biological studies, belongs to ascidians, the main class of tunicates, which are the closest relatives of vertebrates. In Ciona , a project on the ontology of both development and anatomy is ongoing for several years. Its goal is to standardize a resource relating each anatomical structure to developmental stages. Today, the ontology is codified until the hatching larva stage. Here, we present its extension throughout the swimming larva stages, the metamorphosis, until the juvenile stages. For standardizing the developmental ontology, we acquired different time-lapse movies, confocal microscope images and histological serial section images for each developmental event from the hatching larva stage (17.5 h post fertilization) to the juvenile stage (7 days post fertilization). Combining these data, we defined 12 new distinct developmental stages (from Stage 26 to Stage 37), in addition to the previously defined 26 stages, referred to embryonic development. The new stages were grouped into four Periods named: Adhesion, Tail Absorption, Body Axis Rotation, and Juvenile. To build the anatomical ontology, 203 anatomical entities were identified, defined according to the literature, and annotated, taking advantage from the high resolution and the complementary information obtained from confocal microscopy and histology. The ontology describes the anatomical entities in hierarchical levels, from the cell level (cell lineage) to the tissue/organ level. Comparing the number of entities during development, we found two rounds on entity increase: in addition to the one occurring after fertilization, there is a second one during the Body Axis Rotation Period, when juvenile structures appear. Vice versa, one-third of anatomical entities associated with the embryo/larval life were significantly reduced at the beginning of metamorphosis. Data was finally integrated within the web-based resource \"TunicAnatO\", which includes a number of anatomical images and a dictionary with synonyms. This ontology will allow the standardization of data underpinning an accurate annotation of gene expression and the comprehension of mechanisms of differentiation. It will help in understanding the emergence of elaborated structures during both embryogenesis and metamorphosis, shedding light on tissue degeneration and differentiation occurring at metamorphosis.

Insect respiration has long been thought to be solely dependent on an elaborate tracheal system without assistance from the circulatory system or immune cells 1 , 2 . Here we describe that Drosophila crystal cells—myeloid-like immune cells called haemocytes—control respiration by oxygenating Prophenoloxidase 2 (PPO2) proteins. Crystal cells direct the movement of haemocytes between the trachea of the larval body wall and the circulation to collect oxygen. Aided by copper and a neutral pH, oxygen is trapped in the crystalline structures of PPO2 in crystal cells. Conversely, PPO2 crystals can be dissolved when carbonic anhydrase lowers the intracellular pH and then reassembled into crystals in cellulo by adhering to the trachea. Physiologically, larvae lacking crystal cells or PPO2, or those expressing a copper-binding mutant of PPO2, display hypoxic responses under normoxic conditions and are susceptible to hypoxia. These hypoxic phenotypes can be rescued by hyperoxia, expression of arthropod haemocyanin or prevention of larval burrowing activity to expose their respiratory organs. Thus, we propose that insect immune cells collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration. Drosophila haemocytes collaborate with the tracheal system to reserve and transport oxygen through the phase transition of PPO2 crystals, facilitating internal oxygen homeostasis in a process that is comparable to vertebrate respiration.

Antagonistic selection by different predators has been suggested to underlie variation in morphological antipredator traits among and within species. Direct empirical proof is equivocal, however, given the potential interrelationships of morphological and behavioral traits. Here, we tested whether spines in larvae of the dragonfly Leucorrhinia caudalis, which are selected for by fish predators, are selected against by invertebrate aeshnid predators. Using a manipulative approach by cutting spines instead of making comparisons among species or inducing spines, we were able to decouple the presence of spines from other potentially covarying morphological antipredator traits. Results showed survival selection for the loss of spines imposed by invertebrate predation. Moreover, spined and nonspined larval L. caudalis did not differ in the key antipredator behaviors, activity level, and escape burst swimming speed. The observed higher mortality of spined larvae can therefore be directly linked to selection by aeshnid predation against spines.

Amphibian tadpoles display extensive anti-predator phenotypic plasticity, reducing locomotory activity and, with chronic predator exposure, developing relatively smaller trunks and larger tails. In many vertebrates, predator exposure alters activity of the neuroendocrine stress axis. We investigated predator-induced effects on stress hormone production and the mechanistic link to anti-predator defences in Rana sylvatica tadpoles. Whole-body corticosterone (CORT) content was positively correlated with predator biomass in natural ponds. Exposure to caged predators in mesocosms caused a reduction in CORT by 4 hours, but increased CORT after 4 days. Tadpoles chronically exposed to exogenous CORT developed larger tails relative to their trunks, matching morphological changes induced by predator chemical cue; this predator effect was blocked by the corticosteroid biosynthesis inhibitor metyrapone. Tadpole tail explants treated in vitro with CORT increased tissue weight, suggesting that CORT acts directly on the tail. Short-term treatment of tadpoles with CORT increased predation mortality, likely due to increased locomotory activity. However, long-term CORT treatment enhanced survivorship, likely due to induced morphology. Our findings support the hypothesis that tadpole physiological and behavioural/morphological responses to predation are causally interrelated. Tadpoles initially suppress CORT and behaviour to avoid capture, but increase CORT with longer exposure, inducing adaptive phenotypic changes.

In 2004, a new concept was introduced for simplifying identification of larvae of the common nematodes of cattle, sheep and goats that comprises estimates of the lengths of the sheath tail extensions of infective third-stage larvae (L3) of each genus and/or species to that of Trichostrongylus spp., instead of having to be dependent only on measurements in micrometre. For example, if the mean length of the sheath tail extension (the extension of the sheath caudad, beyond the caudal tip of the larva) of Trichostrongylus colubriformis and Trichostrongylus axei is assumed to be ‘X’, then that of Haemonchus contortus is 2.0–2.7 ‘X’ – a difference that is not difficult to estimate. An additional new approach suggested now, particularly for L3 of species and/or genera difficult to differentiate (such as Chabertia ovina and Oesophagostomum columbianum), is to estimate the proportion of the larval sheath tail extension comprising a terminal thin, whip-like filament. For the experienced person, it is seldom necessary to measure more than one or two sheath tail extensions of L3 in a mixed culture, because the identity of most of the remaining L3 can thereafter be estimated in relation to those measured, without having to take further measurements. The aim of this article was to present the novel approach in the form of a working guide for routine use in the laboratory. To facilitate identification, figures and a separate organogram for each of small ruminants and cattle have been added to illustrate the distinguishing features of the common L3.

Habitat is one of the most important factors shaping organismal morphology, but it may vary across life history stages. Ontogenetic shifts in ecology may introduce antagonistic selection that constrains adult phenotype, particularly with ecologically distinct developmental phases such as the free-living, feeding larval stage of many frogs (Lissamphibia: Anura). We test the relative influences of developmental and ecological factors on the diversification of adult skull morphology with a detailed analysis of 15 individual cranial regions across 173 anuran species, representing every extant family. Skull size, adult microhabitat, larval feeding, and ossification timing are all significant factors shaping aspects of cranial evolution in frogs, with late-ossifying elements showing the greatest disparity and fastest evolutionary rates. Size and microhabitat show the strongest effects on cranial shape, and we identify a “large size-wide skull” pattern of anuran, and possibly amphibian, evolutionary allometry. Fossorial and aquatic microhabitats occupy distinct regions of morphospace and display fast evolution and high disparity. Taxa with and without feeding larvae do not notably differ in cranial morphology. However, loss of an actively feeding larval stage is associated with higher evolutionary rates and disparity, suggesting that functional pressures experienced earlier in ontogeny significantly impact adult morphological evolution. The evolution of metamorphic species may be constrained by different ecologies of the larval and adult stages. Here, Bardua et al. show that in frogs, adult ecology is more important than larval ecology for skull evolution, but species that don’t feed as tadpoles evolve faster than those that do.

The Crato Formation (Aptian, Lower Cretaceous) is a fossiliferous deposit of global significance, representing a lacustrine palaeoenvironment which offers insights into aquatic insect taphonomy. Despite its importance, prior studies lacked an actualistic approach. Here, we analyze the preservation of mayflies (Ephemeroptera) and dragonflies (Odonata) from this formation using experimental taphonomy on 253 extant Ephemeroptera and 236 Odonata, alongside 306 fossil specimens. Disarticulation experiments showed that the thorax of modern mayfly larvae disarticulated first, yet Crato Hexagenitidae larvae retained intact thoraces, indicating minimal disturbance and autochthonous deposition. Fossil alate specimens rarely exhibited decay-related wing damage, aligning with short decay times. Dragonfly carcasses exhibited a characteristic leg posture in death, also preserved in Crato fossils, further suggesting minimal transport. Additionally, fossil dragonflies retained labial masks, the first structure to disarticulate experimentally, consistent with parautochthonous assemblages. Mayfly larvae exposed to low salinity during experiments exhibited excessive defecation before death, hinting at possible low salinity conditions in the Crato palaeoenvironment, though preservational challenges obscure confirmation. During experimentation, we also noticed that all carcasses immediately floated under hypersaline conditions, while carcasses immersed in non-hypersaline conditions went through slower decomposition. Thus, we can safely propose with experimental data that microbial biofilms on the surface of the water were acting during carcass sinking in this deposit.