Pleuropodia are limb-derived glandular organs that transiently appear on the first abdominal segment in embryos of insects from majority of “orders”. They are missing in the genetic model Drosophila and little is known about them. Experiments carried out on orthopteran insects 80 years ago indicated that the pleuropodia secrete a “hatching enzyme” that digests the serosal cuticle to enable the larva to hatch, but evidence by state-of-the-art molecular methods is missing. We used high-throughput RNA-sequencing to identify the genes expressed in the pleuropodia of the locust Schistocerca gregaria (Orthoptera). First, using transmission electron microscopy we studied the development of the pleuropodia during 11 stages of the locust embryogenesis. We show that the glandular cells differentiate and start secreting just before the definitive dorsal closure of the embryo and the secretion granules outside the cells become more abundant prior to hatching. Next, we generated a comprehensive embryonic reference transcriptome for the locust and used it to study genome wide gene expression across ten morphologicaly defined stages of the pleuropodia. We show that when the pleuropodia have morphological markers of functional organs and produce secretion, they are primarily enriched in transcripts associated with transport functions. They express genes encoding enzymes capable of digesting cuticular protein and chitin. These include the potent cuticulo-lytic Chitinase 5, whose transcript rises just before hatching. Unexpected finding was the enrichment in transcripts for immunity-related enzymes. This indicates that the pleuropodia are equipped with epithelial immunity similarly as barrier epithelia in postembryonic stages. These data provide transcriptomic support for the historic hypothesis that pleuropodia produce cuticle-degrading enzymes and function in hatching. They may also have other functions, such as facilitation of embryonic immune defense. By the genes that they express the pleuropodia are specialized embryonic organs and apparently an important though neglected part of insect physiology.

Amphibians are one of the most susceptible groups to climate change as their development occurs in aquatic environments or in microhabitats with high humidity. Accordingly, our primary objective was to investigate the chronic physiological responses seen in early larval to adult stages of Polypedates cruciger (Common hourglass tree frog) to future climate change based on continuous exposure to elevated temperature and elevated CO2 -induced low water pH. Free-swimming and free-feeding tadpoles were observed until metamorphosis under four experimental treatments; two elevated temperatures, one elevated CO2 (reduced pH) and a control maintained at ambient temperature (29 °C ± 1 °C) and CO2 (pH = 7). Elevated temperature treatments were maintained at 32 °C ± 0.5 °C and 34 °C ± 0.5 °C to represent respectively, the future climate scenarios RCP2.6 (Representative Concentration Pathway 2.6, the ‘base-case’ scenario) and RCP8.5 (‘business-as-usual’ scenario) according to the 5th Assessment Report of the IPCC. Elevated CO2 treatment was maintained within the pH range of 5.5–5.6 representing the range expected between RCP8.5 and RCP2.6. Compared to the control, elevated CO2 accelerated phenological progression of tadpoles through Gosner stages, thus resulting in lower body size at metamorphosis. Both elevated temperatures significantly delayed the development and reduced the growth of tadpoles. 100% mortality was observed in 34 °C treatment before metamorphosis (before Gosner stage 36) while all the tadpoles died after metamorphosis (at Gosner stage 46) in 32 °C treatment. Elevated CO2 increased tadpole activity, in terms of their swimming speed, while both of the elevated temperatures reduced it compared to the control. Catalase activity increased at elevated CO2. Ammonia excretion by tadpoles was decreased by elevated CO2, but increased under temperature elevation. Both Elevated CO2 and temperature treatments reduced the white blood cell count and its percentage of thrombocytes. Percentages of lymphocytes, monocytes and neutrophils were increased at 32 °C, while lymphocyte percentage and lysozyme activity were increased at elevated CO2. Several deformities were observed in tadpoles at elevated temperature and CO2. Elevated temperatures and reduced pH due to elevated CO2, being major features of climate change, increase the vulnerability of amphibians, who are already one of the most threatened vertebrate groups. Based on our observations on the model amphibian species P. cruciger, increased vulnerability to climate change occurs by reducing their growth, body size and motility while also reducing their immunity and inducing physical deformities. These impacts are highly-likely to reduce the foraging, competitive and reproductive capabilities in their natural habitats. We conclude further that even the ‘best-case’ scenario of future climate change can impose significant physiological impacts that could threaten amphibian populations on broader spatial and temporal scales.

Based on 20,000 records representing c. 11,000 individuals from an 8-year capture-mark-recapture (CMR) study, we tested and confirmed a new case of invariant clutch size (ICS) in a sexually dichromatic lacertid lizard, Takydromus viridipunctatus. In the grassland habitat of the early succession stage, females showed strictly low and invariant clutch size, multiple clutches in a breeding season, high reproductive potential, and annual breeding cycles that correspond to the emergence of male courtship coloration. The hatchlings mature quickly, and join the adult cohort for breeding within a few months, whereas adults show low survival rates and a short lifespan, such that most die within one year. Mortality increased in both sexes during the breeding season, especially in females, indicating an unequal cost of reproduction in survival. These life history characters may be explained by two non-exclusive hypotheses of ICS—arboreal hypothesis and predation hypothesis—within the ecological context of their habitat. Our study highlights a confirmed case of ICS, which adapts well to this r-selected grassland habitat that experiences seasonal fluctuation and frequent disturbance.

The apical organ is the most prominent neural structure in spiralian larvae. Although it has been thoroughly investigated in larvae of the class Pilidiophora in phylum Nemertea, studies on its structure in other nemertean larvae are limited. Most adult hoplonemertean worms have a frontal organ located in a position corresponding to that of the larval apical organ. The development and sensory function of the frontal organ has not been thoroughly characterized to date. The apical organ in the early rudiment stage of Quasitetrastemma stimpsoni larvae consists of an apical plate enclosed by ducts of frontal gland cells and eight apical neurons. The apical plate is abundantly innervated by neurites of apical neurons. During the late rudiment stage, the larval apical organ has external innervation from below by two subapical-plate neurons, along with 11 apical neurons, and its plate contains serotonin-like immunoreactive (5-HT-lir) cells. In the vermicular stage (free-swimming juvenile), the number of apical neurons is reduced, and their processes are resorbed. Serotonin is detected in the apical plate with no visible connection to apical neurons. In adult worms, the frontal organ has a small apical pit with openings for the frontal gland ducts. The organ consists of 8 to 10 densely packed 5-HT-lir cells that form the roundish pit. Although the ultrastructure of the Q. stimpsoni larval apical organ closely resembles that of the apical organ of Polycladida larvae, the former differs in the presence of flask-shaped neurons typical of Spiralia. Significant differences in the structure of the apical organs of hoplonemertean and pilidia larvae point to two different paths in the evolutionary transformation of the ancestral apical organ. Ultrastructural and immunoreactive analyses of the apical organ of a hoplonemertean larva in the late rudiment and vermicular stages and the frontal organ of the adult worms identified common morphological and functional features. Thus, we hypothesize that the larval apical organ is modified during morphogenesis to form the adult frontal organ, which fulfills a sensory function in the hoplonemertean worm. This unique developmental trait distinguishes the Hoplonemertea from other nemertean groups.

In animals with altricial offspring, most growth occurs after birth and may be optimized by post-natal maternal care. Maternal effects on growth may be influenced by individual characteristics of the mothers, such as social status, individual investment strategies and the length of association with offspring. The prolonged juvenile dependence seen in humans is a distinctive life history adaptation, which may have evolved to facilitate sustained somatic and brain growth. In chimpanzees, offspring are typically weaned at approximately 4 years old, yet immature individuals continue to associate with their mothers for up to 10 years beyond weaning. Whether this lengthy association or the individual characteristics of mothers influences growth patterns in this species is not clear. The relationship between urinary creatinine and specific gravity is an established non-invasive measure of muscle mass in humans and chimpanzees. We analysed the urinary creatinine and specific gravity of 1318 urine samples from 70 wild chimpanzees from the Taï Forest, Ivory Coast aged 4 to 15 years. We showed a clear increase in urinary creatinine levels with age in both males and females, replicating established growth curves in this species and reaffirming this measure as a reliable proxy for lean body mass. Comparing those who experience maternal loss (orphans) with non-orphan chimpanzees, maternal presence beyond weaning age and into late juvenility positively influenced offspring muscle mass throughout ontogeny such that orphans had significantly less muscle mass than age-matched non-orphans. In age-matched offspring with mothers, those with high-ranking mothers had greater muscle mass. Accounting for variation in muscle mass attributable to maternal presence, we found no effect of maternal investment (length of inter birth interval, from own birth to birth of following sibling) on offspring muscle mass. Chimpanzee mothers have an extended and multi-faceted influence on offspring phenotypes. Our results suggest that maternal investment extends beyond lactation and into early adulthood and has clear benefits to offspring physical development. Therefore, prolonged juvenile dependence, although unique in its form in human societies, may be a trait with deeper evolutionary origins.

Current patterns of biological distribution result from the deep past. Of particular interest, some closely related species appear at high latitudes of both hemispheres, but not in between, a pattern known as antitropical distribution. However, the timing, pathway, and drivers of antitropical distributions remain mostly unknown. Here we describe a new fossil, a left tympanic bulla (part of the ear bones), from the Middle/Late Pleistocene (0.78–0.01 mya, but not excluding the possibility of Holocene in age, as the specimen was dredged from the sea bottom and the geological horizon remains uncertain) of Taiwan. The tympanic bulla is diagnostic in baleen whales, and this specimen shows morphological features that are identical to extant Eubalaena, including: relatively large size (the anteroposterior length is 117 mm); rectangular outline in medial view; short anterior lobe, judging from the remaining of the lateral furrow; squared anterior margin; prominent transverse crease on the involucrum; transversely compressed in anterior view; well-developed and rounded outer lip; and parallel involucral and main ridges. Although incomplete, the morphological characters and overall similarity to extant Eubalaena allow a reliable taxonomic assignment to Eubalaena sp. The occurrence of a Pleistocene Eubalaena on the southern margin of the western North Pacific is the first balaenid fossil evidence indicative of the biotic interchange between two hemispheres leading to the origin of antitropical distribution in the Pleistocene; alternatively, this specimen might merely represent an extra-limital record of the North Pacific Eubalaena. Furthermore, this find suggests that the Eubalaena interchange, being one of the largest species displaying antitropical distribution pairs in the history of life, likely took place along the western Pacific. Notably, this does not preclude the Eubalaena interchange from other routes, such as the eastern Pacific or the Atlantic Ocean, and future finds should test the scenario for the biotic interchange between Northern and Southern Hemispheres of Eubalaena.

Despite numerous approaches to the resolution of euarthropod phylogeny, mainly based on modern sequence information and traditional external morphology, the resulting hypotheses are often contradictory and leave many questions about euarthropod evolution unanswered. The comparison of developmental and structural aspects of the nervous system has shown to be a valuable contribution to the assessment of current phylogenetic hypotheses. One promising approach for the generation of new character sets is the morphology of transmitter systems and the discovery of individually identifiable neurons, which allow phylogenetic comparisons on the single cell level. In this context, the serotonin transmitter system has been investigated to a considerable degree. Studies to date have yielded important stimuli to our understanding of euarthropod relationships and the evolution of their nervous systems. However, data on other transmitter systems remain fragmented, and their value with respect to phylogenetic questions remains speculative. The biogenic amine histamine is a promising transmitter; a substantial amount of data has been reported in the literature and the homology of some histaminergic neurons has been suggested. Here, we present a comprehensive review of histaminergic neurons in the ventral nerve cord of Euarthropoda. Using immunocytochemical labeling of histamine combined with confocal laser-scanning microscopy, we investigated the transmitter system in phylogenetically relevant taxa, such as Zygentoma, Remipedia, Diplopoda, and Arachnida. By reconstructing ground patterns, we evaluated the significance of this specific character set for euarthropod phylogeny. With this approach, we identified a set of neurons, which can be considered homologous within the respective major taxon. In conclusion, the histaminergic system contains useful information for our understanding of euarthropod phylogeny, supporting the proposed clades Tetraconata and Mandibulata. Furthermore, this character set has considerable potential to help resolve relationships within the major clades at a deeper level of taxonomy, due to the considerable variability in neurite morphology.

The geometric patterns that adorn the shells of many phylogenetically disparate molluscan species are comprised of pigments that span the visible spectrum. Although early chemical studies implicated melanin as a commonly employed pigment, surprisingly little evidence generated with more recent and sensitive techniques exists to support these observations. Here we present the first mass spectrometric investigations for the presence of eumelanin and pheomelanin in 13 different molluscan species from three conchiferan classes: Bivalvia, Cephalopoda and Gastropoda. In the bivalve Mytilus edulis we demonstrate that eumelanin mainly occurs in the outermost, non-mineralised and highly pigmented layer of the shell (often referred to as the periostracum). We also identified eumelanin in the shells of the cephalopod Nautilus pompilius and the marine gastropods Clanculus pharaonius and Steromphala adriatica. In the terrestrial gastropod Cepaea nemoralis we verify the presence of pheomelanin in a mollusc shell for the first time. Surprisingly, in a large number of brown/black coloured shells we did not find any evidence for either type of melanin. We recommend methods such as high-performance liquid chromatography with mass spectrometric detection for the analysis of complex biological samples to avoid potential false-positive identification of melanin. Our results imply that many molluscan species employ as yet unidentified pigments to pattern their shells. This has implications for our understanding of how molluscs evolved the ability to pigment and pattern their shells, and for the identification of the molecular mechanisms that regulate these processes.

Self-incompatibility, fusion/non-fusion reactions, and contact reactions (CRs) have all been identified as allorecognition phenomena in ascidians. CR is a reaction characteristic of the hemocytes of Halocynthia roretzi, whereby they release phenol oxidase (PO) upon contact with non-self hemocytes. Thus, these cells may represent a primitive form of the vertebrate immune system. In the present study, we focused on the CR of H. roretzi hemocytes and sought to identify self-marker proteins that distinguish between self and non-self cells. We initially generated a CR-inducing monoclonal antibody against the complete hemocyte membrane-protein complement (mAb11B16B10). This antibody was identified based on the differential induction of PO activity in individual organisms. The level of PO activity induced by this antibody in individual ascidians was consistent with the observed CR-induced PO activity. mAb11B16B10 recognized a series of 12 spots corresponding to a 100-kDa protein, with differing isoelectric points (pIs). A comparison of the 2D electrophoresis gels of samples from CR-reactive/non-reactive individuals revealed that some spots in this series in hemocytes were common to the CR-non-inducible individuals, but not to CR-inducible individuals. We cloned the corresponding gene and named it Halocynthia roretzi self-marker-like protein-1 (HrSMLP1). This gene is similar to the glycoprotein DD3–3 found in Dictyostelium, and is conserved in invertebrates. We generated a CR-inducing monoclonal antibody (mAb11B16B10) that recognized a series of novel membrane proteins (HrSMLP1) in the hemocytes of H. roretzi. The combination of expressed spots of HrSMLP1 distinguishes non-self cells from self cells with respect to CR inducibility. Given that the HrSMLP1 gene is a single gene, it may represent a novel type of self-marker protein with a role in CR.

Absorption spectra of opsin-based pigments are tuned from the UV to the red regions by interactions of the chromophore with surrounding amino acid residues. Both vertebrates and invertebrates possess long-wavelength-sensitive (LWS) opsins, which underlie color vision involving “red” sensing. The LWS opsins have independently evolved in each lineage, which suggests the existence of diverse mechanisms in spectral tuning. In vertebrate LWS opsins, the mechanisms underlying spectral tuning have been well characterized by spectroscopic analyses with recombinant pigments of wild type (WT) and mutant opsins. However in invertebrate LWS opsins including insect ones, the mechanisms are largely unknown due to the difficulty in obtaining recombinant pigments. Here we have overcome the problem by analyzing heterologous action spectra based on light-dependent changes in the second messenger in opsin-expressing cultured cells. We found that WTs of two LWS opsins of the butterfly, Papilio xuthus, PxRh3 and PxRh1 have the wavelengths of the absorption maxima at around 570 nm and 540 nm, respectively. Analysis of a series of chimeric mutants showed that helix III is crucial to generating a difference of about 15 nm in the wavelength of absorption maxima of these LWS opsins. Further site-directed mutations in helix III revealed that amino acid residues at position 116 and 120 (bovine rhodopsin numbering system) are involved in the spectral tuning of PxRh1 and PxRh3, suggesting a different spectral tuning mechanism from that of primate LWS opsins.

Brachyuran crabs can effectively modulate cardiac stroke volume independently of heart rate in response to abiotic drivers. Non-invasive techniques can help to improve the understanding of cardiac performance parameters of these animals. This study demonstrates the in vivo quantification of cardiac performance parameters through magnetic resonance imaging (MRI) on the edible crab Cancer pagurus. Furthermore, the suitability of signal integrals of infra-red photoplethysmographs as a qualitative tool is assessed under severe hypoxia. Multi-slice self-gated cardiac cinematic (CINE) MRI revealed the structure and motion of the ventricle to quantify heart rates, end-diastolic volume, end-systolic volume, stroke volume and ejection fraction. CINE MRI showed that stroke volumes increased under hypoxia because of a reduction of end-systolic volumes at constant end-diastolic volumes. Plethysmograph recordings allowed for automated heart rate measurements but determination of a qualitative stroke volume proxy strongly depended on the position of the sensor on the animal. Both techniques revealed a doubling in stroke volumes after 6 h under severe hypoxia (water PO2 = 15% air saturation). MRI has allowed for detailed descriptions of cardiac performance in intact animals under hypoxia. The temporal resolution of quantitative non-invasive CINE MRI is limited but should encourage further refining. The stroke volume proxy based on plethysmograph recordings is feasible to complement other cardiac measurements over time. The presented methods allow for non-destructive in vivo determinations of multiple cardiac performance parameters, with the possibility to study neuro-hormonal or environmental effects on decapod cardio physiology.

Acoustic signals are used by many animals to transmit information. Variation in the acoustic characteristics of these signals often covaries with morphology and can relay information about an individual’s fitness, sex, species, and/or other characteristics important for both mating and defense. Tiger moths (Lepidoptera: Erebidae: Arctiinae) use modified cuticular plates called “tymbal organs” to produce ultrasonic clicks which can aposematically signal their toxicity, mimic the signals of other species, or, in some cases, disrupt bat echolocation. The morphology of the tymbal organs and the sounds they produce vary greatly between species, but it is unclear how the variation in morphology gives rise to the variation in acoustic characteristics. This is the first study to determine how the morphological features of tymbals can predict the acoustic characteristics of the signals they produce. We show that the number of striations on the tymbal surface (historically known as “microtymbals”) and, to a lesser extent, the ratio of the projected surface area of the tymbal to that of the thorax have a strong, positive correlation with the number of clicks a moth produces per unit time. We also found that some clades have significantly different regression coefficients, and thus the relationship between microtymbals and click rate is also dependent on the shared ancestry of different species. Our predictive model allows the click rates of moths to be estimated using preserved material (e.g., from museums) in cases where live specimens are unavailable. This has the potential to greatly accelerate our understanding of the distribution of sound production and acoustic anti-bat strategies employed by tiger moths. Such knowledge will generate new insights into the evolutionary history of tiger moth anti-predator defenses on a global scale.

The origin of birds is marked by a significant decrease in body size along with an increase in relative forelimb size. However, before the evolution of flight, both traits may have already been related: It has been proposed that an evolutionary trend of negative forelimb allometry existed in non-avian Theropoda, such that larger species often have relatively shorter forelimbs. Nevertheless, several exceptions exist, calling for rigorous phylogenetic statistical testing. Here, we re-assessed allometric patterns in the evolution of non-avian theropods, for the first time taking into account the non-independence among related species due to shared evolutionary history. We confirmed a main evolutionary trend of negative forelimb allometry for non-avian Theropoda, but also found support that some specific subclades (Coelophysoidea, Ornithomimosauria, and Oviraptorosauria) exhibit allometric trends that are closer to isometry, losing the ancestral negative forelimb allometry present in Theropoda as a whole. Explanations for negative forelimb allometry in the evolution of non-avian theropods have not been discussed, yet evolutionary allometric trends often reflect ontogenetic allometries, which suggests negative allometry of the forelimb in the ontogeny of most non-avian theropods. In modern birds, allometric growth of the limbs is related to locomotor and behavioral changes along ontogeny. After reviewing the evidence for such changes during the ontogeny of non-avian dinosaurs, we propose that proportionally longer arms of juveniles became adult traits in the small-sized and paedomorphic Aves.

Amphibians have evolved a remarkable diversity of defensive mechanisms against predators. One of the most conspicuous components in their defense is related to their ability to produce and store a high variety of bioactive (noxious to poisonous) substances in specialized skin glands. Previous studies have shown that T. verrucosus is poisonous with the potential to truly harm or even kill would-be predators by the effect of its toxic skin secretions. However, little is known on form and function of the skin glands responsible for production and release of these secretions. By using light- and scanning electron microscopy along with confocal laser scanning microscopy, we show that T. verrucosus exhibits three different multicellular skin glands: one mucous- and two granular glands. While mucous glands are responsible for the production of the slippery mucus, granular glands are considered the production site of toxins. The first type of granular glands (GG1) is found throughout the skin, though its average size can vary between body regions. The second type of granular glands (GG2) can reach larger dimensions compared with the former type and is restricted to the tail region. Despite their different morphology, all three skin gland types are enwrapped by a distinct myoepithelial sheath that is more prominently developed in the granular (i.e. poison-) glands compared to the mucous glands. The myoepithelial sheath consists of one layer of regularly arranged slender myoepithelial cells that run from the gland pore to the basal gland pole. This study shows that the skin in the Himalayan newt T. verrucosus displays one mucus- and two poison gland types enwrapped by a myoepithelial sheath. Contraction of the myoepithelium squeezes the glands and glandular content is released upon the skin surface where the secretion can deploy its defensive potential.

DNA transposons are generally destroyed by mutations and have short lifespans in hosts, as they are neutral or harmful to the host and therefore not conserved by natural selection. The clawed frog Xenopus harbors many DNA transposons and certain families, such as T2-MITE, have extremely long lives. These have ancient origins, but have shown recent transposition activity. In addition, certain transposase genes may have been “domesticated” by Xenopus and conserved over long time periods by natural selection. The aim of this study was to elucidate the evolutionary interactions between the host and the long-lived DNA transposon family it contains. Here, we investigated the molecular evolution of the Kolobok DNA transposon superfamily. Kolobok is thought to contribute to T2-MITE transposition. In the diploid western clawed frog Xenopus tropicalis and the allotetraploid African clawed frog Xenopus laevis, we searched for transposase genes homologous to those in the Kolobok superfamily. To determine the amplification and domestication of these genes, we used molecular phylogenetics and analyses of copy numbers, conserved motifs, orthologous gene synteny, and coding sequence divergence between the orthologs of X. laevis and X. tropicalis, or between those of two distant X. tropicalis lineages. Among 38 X. tropicalis and 24 X. laevis prospective transposase genes, 10 or more in X. tropicalis and 14 or more in X. laevis were apparently domesticated. These genes may have undergone multiple independent domestications from before the divergence of X. laevis and X. tropicalis. In contrast, certain other transposases may have retained catalytic activity required for transposition and could therefore have been recently amplified. Multiple domestication of certain transposases and prolonged conservation of the catalytic activity in others suggest that Kolobok superfamily transposons were involved in complex, mutually beneficial relationships with their Xenopus hosts. Some transposases may serve to activate long-lived T2-MITE subfamilies.

The skeletal musculature of gnathostomes, which is derived from embryonic somites, consists of epaxial and hypaxial portions. Some hypaxial muscles, such as tongue and limb muscles, undergo de-epithelialization and migration during development. Delamination and migration of these myoblasts, or migratory muscle precursors (MMPs), is generally thought to be regulated by hepatocyte growth factor (HGF) and receptor tyrosine kinase (MET) signaling. However, the prevalence of this mechanism and the expression patterns of the genes involved in MMP development across different vertebrate species remain elusive. We performed a comparative analysis of Hgf and Met gene expression in several vertebrates, including mouse, chicken, dogfish (Scyliorhinus torazame), and lamprey (Lethenteron camtschaticum). While both Hgf and Met were expressed during development in the mouse tongue muscle, and in limb muscle formation in the mouse and chicken, we found no clear evidence for the involvement of HGF/MET signaling in MMP development in shark or lamprey embryos. Our results indicate that the expressions and functions of both Hgf and Met genes do not represent shared features of vertebrate MMPs, suggesting a stepwise participation of HGF/MET signaling in MMP development during vertebrate evolution.

Tyramine, known as a “trace amine” in mammals, modulates a wide range of behavior in invertebrates; however, the underlying cellular and circuit mechanisms are not well understood. In the nematode Caenorhabditis elegans (C. elegans), tyramine affects key behaviors, including foraging, feeding, and escape responses. The touch-evoked backward escape response is often coupled with a sharp omega turn that allows the animal to navigate away in the opposite direction. Previous studies have showed that a metabotropic tyramine receptor, SER-2, in GABAergic body motor neurons controls deep body bending in omega turns. In this study, we focused on the role of tyramine in GABAergic head motor neurons. Our goal is to understand the mechanism by which tyraminergic signaling alters neural circuit activity to control escape behavior. Using calcium imaging in freely moving C. elegans, we found that GABAergic RME motor neurons in the head had high calcium levels during forward locomotion but low calcium levels during spontaneous and evoked backward locomotion. This calcium decrease was also observed during the omega turn. Mutant analyses showed that tbh-1 mutants lacking only octopamine had normal calcium responses, whereas tdc-1 mutants lacking both tyramine and octopamine did not exhibit the calcium decrease in RME. This neuromodulation was mediated by SER-2. Moreover, tyraminergic RIM neuron activity was negatively correlated with RME activity in the directional switch from forward to backward locomotion. These results indicate that tyramine released from RIM inhibits RME via SER-2 signaling. The omega turn is initiated by a sharp head bend when the animal reinitiates forward movement. Interestingly, ser-2 mutants exhibited shallow head bends and often failed to execute deep-angle omega turns. The behavioral defect and the abnormal calcium response in ser-2 mutants could be rescued by SER-2 expression in RME. These results suggest that tyraminergic inhibition of RME is involved in the control of omega turns. We demonstrate that endogenous tyramine downregulates calcium levels in GABAergic RME motor neurons in the head via the tyramine receptor SER-2 during backward locomotion and omega turns. Our data suggest that this neuromodulation allows deep head bending during omega turns and plays a role in the escape behavior in C. elegans.

Ticks can survive long periods without feeding but, when feeding, ingest large quantities of blood, resulting in a more than 100-fold increase of body volume. We study morphological adaptations to changes in opisthosoma volume during feeding in the castor bean tick, Ixodes ricinus. We aim to understand the functional morphological features that accommodate enormous changes in volume changes. Using light and electron microscopy, we compare the cuticle and epidermis of the alloscutum, the epithelium of the midgut diverticula, and the tracheae of adult female ticks when fasting, semi-engorged, and fully engorged. Our results add to an existing body of knowledge that the area of the epidermis increases by cellular differentiation, cellular hypertrophy, and changes in the shape of epithelial cells from pseudostratified to single layered prismatic in semi-engorged ticks, and to thin squamous epithelium in fully engorged ticks. We did not find evidence for cell proliferation. The midgut diverticula accommodate the volume increase by cellular hypertrophy and changes in cell shape. In fully engorged ticks, the epithelial cells of the midgut diverticula are stretched to an extremely thin, squamous epithelium. Changes in size and shape (and cell divisions) contribute to the accommodation of volume changes. Tracheae do not increase in size, but extend in length, thus following the volume changes of the opisthosoma in feeding ticks to secure oxygen supply to the internal organs. Changes of epithelial tissue configuration in the epidermis and the midgut diverticula are described as important components of the morphological response to feeding in ticks. We provide evidence for a previously unknown mechanism hosted in the endocuticle of the tracheae that allows the tracheae of castor bean ticks to expand when the body volume increases and the distance between the respiratory spiracle and the oxygen demanding tissue enlarges. This is the first report of expandable tracheae in arthropods.

Geographical patterns and degrees of genetic divergence among populations differ between species, reflecting relative potentials for speciation or cladogenesis and differing capacities for environmental adaptation. Identification of factors that contribute to genetic divergence among populations is important to the understanding of why some species exhibit greater interpopulation genetic divergence. In this study, we calculated the mean pairwise genetic distances among populations as species’ average genetic divergence by a phylogeny using nuclear and mitochondrial genes of 303 individuals from 33 Cuban Anolis species and estimated species ages by another phylogeny using nuclear and mitochondrial genes of 51 Cuban and 47 non-Cuban Anolis species. We identified factors that influence species’ differences in genetic divergence among 26 species of Anolis lizards from Cuba. Species ages, environmental heterogeneity within species ranges, and ecomorph types were considered as factors affecting average genetic divergences among populations. The phylogenies presented in this study provide the most comprehensive sampling of Cuban Anolis species to date. The phylogeny showed more conservative evolution of Anolis ecomorphs within Cuba and identified twig anoles as a monophyletic group. Subsequent Phylogenetic Generalized Least Squares (PGLS) analyses showed that species age was positively correlated with species’ average genetic divergence among populations. Although previous studies have focused on factors affecting genetic divergence within species, the present study showed for the first time that species differences in genetic divergence could be largely affected by species age.

The terrestrial slug Limax has long been used as a model for the study of olfactory information processing and odor learning. Olfactory inputs from the olfactory epithelium are processed in the tentacular ganglion and then in the procerebrum. Glutamate and acetylcholine are the major neurotransmitters used in the procerebrum. Phe-Met-Arg-Phe-NH2 (FMRFamide) has been shown to be involved in the regulation of the network activity of the procerebrum. Although there are thought to be various RFamide family peptides other than FMRFamide that are potentially recognized by anti-FMRFamide antibody in the central nervous system of mollusks, identifying the entire repertoire of RFamide peptides in Limax has yet to be achieved. In the present study, we made a comprehensive search for RFamide peptide-encoding genes from the transcriptome data of Limax, and identified 12 genes. The expression maps of these RFamide genes were constructed by in situ hybridization in the cerebral ganglia including the procerebrum, and in the superior/inferior tentacles. Ten of 12 genes were expressed in the procerebrum, and nine of 12 genes were expressed in the tentacular ganglia. Gly-Ser-Leu-Phe-Arg-Phe-NH2 (GSLFRFamide), which is encoded by two different genes, LFRFamide1 (Leu-Phe-Arg-Phe-NH2–1) and LFRFamide2 (Leu-Phe-Arg-Phe-NH2–2), decreased the oscillatory frequency of the local field potential oscillation in the procerebrum when exogenously applied in vitro. We also found by immunohistochemistry that the neurons expressing pedal peptide send efferent projections from the procerebrum back to the tentacular ganglion. Our findings suggest the involvement of a far wider variety of RFamide family peptides in the olfactory information processing in Limax than previously thought.

Mudskippers are amphibious fishes that use their pectoral fins to move on land. Their pectoral fins are specifically modified for terrestrial locomotion. Studies of the anatomy and kinematics of adult mudskippers suggest that modifications of the pectoral fins, such as their protrusion and elongation of the proximal radials, may provide greater control and flexibility in pectoral fin–based locomotion. However, it is unknown when and how the unique features of these pectoral fins form during the development of mudskippers, which begin life as a planktonic organism. Here we examined the developmental process of the pectoral fins of the mudskipper Periophthalmus modestus to address these questions. We also observed other developmental characteristics to provide clarified descriptions, including indicative morphological changes that occur during metamorphosis. Our results show that the localized cell division of the proximal part of the endoskeletal disc—the primordium of the proximal radials—and subsequent cell division along the proximal-distal axis, which is restricted to the distal part of the disc during the larva-to-juvenile transition (metamorphosis), lead to the elongation of the proximal radials.

Planarians are non-parasitic Platyhelminthes (flatworms) famous for their regeneration ability and for having a well-organized brain. Dugesia japonica is a typical planarian species that is widely distributed in the East Asia. Extensive cellular and molecular experimental methods have been developed to identify the functions of thousands of genes in this species, making this planarian a good experimental model for regeneration biology and neurobiology. However, no genome-level information is available for D. japonica, and few gene regulatory networks have been identified thus far. To obtain whole-genome information on this species and to study its gene regulatory networks, we extracted genomic DNA from 200 planarians derived from a laboratory-bred asexual clonal strain, and sequenced 476 Gb of data by second-generation sequencing. Kmer frequency graphing and fosmid sequence analysis indicated a complex genome that would be difficult to assemble using second-generation sequencing short reads. To address this challenge, we developed a new assembly strategy and improved the de novo genome assembly, producing a 1.56 Gb genome sequence (DjGenome ver1.0, including 202,925 scaffolds and N50 length 27,741 bp) that covers 99.4% of all 19,543 genes in the assembled transcriptome, although the genome is fragmented as 80% of the genome consists of repeated sequences (genomic frequency ≥ 2). By genome comparison between two planarian genera, we identified conserved non-coding elements (CNEs), which are indicative of gene regulatory elements. Transgenic experiments using Xenopus laevis indicated that one of the CNEs in the Djndk gene may be a regulatory element, suggesting that the regulation of the ndk gene and the brain formation mechanism may be conserved between vertebrates and invertebrates. This draft genome and CNE analysis will contribute to resolving gene regulatory networks in planarians. The genome database is available at: http://www.planarian.jp .

Following publication of the original article [1] the author advised that (in the ‘Discussion’ section) the letter ‘L’ and ‘D’ in some expressions (namely, D and L in L-Trp, D-Trp and L-Asn-D-Trp-L-Phe) were not written in the format of ‘small capital letters’. Rather, they were written as lower-case letters.

Entrainment to the environmental light cycle is an essential property of the circadian clock. Although the compound eye is known to be the major photoreceptor necessary for entrainment in many insects, the molecular mechanisms of photic entrainment remain to be explored. We found that cryptochromes (crys) and c-fos mediate photic entrainment of the circadian clock in a hemimetabolous insect, the cricket Gryllus bimaculatus. We examined the effects of RNA interference (RNAi)-mediated knockdown of the cry genes, Gb’cry1 and Gb’cry2, on photic entrainment, and light-induced resetting of the circadian locomotor rhythm. Gb’cry2 RNAi accelerated entrainment for delay shifts, while Gb’cry1/ Gb’cry2 double RNAi resulted in significant lengthening of transient cycles in both advance and delay shifts, and even in entrainment failure in some crickets. Double RNAi also strongly suppressed light induced resetting. The Gb’cry-mediated phase shift or resetting of the rhythm was preceded by light-induced Gb’c-fosB expression. We also found that Gb’c-fosB, Gb’cry2 and Gb’period (Gb’per) were likely co-expressed in some optic lobe neurons. Based on these results, we propose a novel model for photic entrainment of the insect circadian clock, which relies on the light information perceived by the compound eye.

While skeletal evolution has been extensively studied, the evolution of limb muscles and brachial plexus has received less attention. In this review, we focus on the tempo and mode of evolution of forelimb muscles in the vertebrate history, and on the developmental mechanisms that have affected the evolution of their morphology. Tetrapod limb muscles develop from diffuse migrating cells derived from dermomyotomes, and the limb-innervating nerves lose their segmental patterns to form the brachial plexus distally. Despite such seemingly disorganized developmental processes, limb muscle homology has been highly conserved in tetrapod evolution, with the apparent exception of the mammalian diaphragm. The limb mesenchyme of lateral plate mesoderm likely plays a pivotal role in the subdivision of the myogenic cell population into individual muscles through the formation of interstitial muscle connective tissues. Interactions with tendons and motoneuron axons are involved in the early and late phases of limb muscle morphogenesis, respectively. The mechanism underlying the recurrent generation of limb muscle homology likely resides in these developmental processes, which should be studied from an evolutionary perspective in the future.

The developmental anatomy of the dorsal hindbrain in an elasmobranch fish, Leucoraja erinacea, is described. We focus on the cerebellum, which is a synapomorphy for gnathostomes. Cerebellar development in L. erinacea, a representative of the most basal gnathostome lineage, may be a proxy for the ancestral state of cerebellar development. We also focus on sensory processing regions termed ‘cerebellum-like’ structures due to common anatomical and physiological features with the cerebellum. These structures may be considered generatively homologous if they share common developmental features. To test this hypothesis, the morphological development of the cerebellum and cerebellum-like structures must first be described. Of particular importance is the development of common features, such as the molecular layer, which is the defining characteristic of these structures. The molecular layers of the cerebellum and cerebellum-like structures are supplied with parallel fiber axons from distinct granule cell populations. These are the lateral granule mass, the dorsal granular ridge, the medial granule mass, and the granular eminences of the cerebellum. Cerebellar and cerebellar-like development in L. erinacea is similar to development in other elasmobranchs. The temporal order in which these granule cell populations develop suggests an evolutionary history of duplication or expansion of an existing developmental event.

The study of chondrocrania has a long tradition with a focus on single specimens and stages. It revealed great interspecific diversity and a notion of intraspecific variation. As an embryonic structure, the chondrocranium is subject to major changes in ontogeny with resorption and ossification of different cartilaginous structures. The cupula nasi anterior is the anteriormost portion of the cartilaginous nasal capsule and is expected to mirror much of the animal's life history and lifestyle. Its diversity in mammals is reflected in the external nasal anatomy of newborns. Marsupials and placentals show marked differences, likely related to breathing and suckling behavior. We examined histological sections of five marsupial and three placentals species and traced the development of the cupula nasi anterior and the anterior nasal capsule. We found ontogenetic variation for nearly 50% of the 43 characters defined herein. By comparing to the literature and considering ontogenetic variation, we performed an analysis of character evolution in 70 mammalian species and reconstructed the nasal anatomy of the therian ancestor. At birth, marsupials have a complete but simple cupula nasi anterior, whereas placentals display a more diverse morphology due to reductions and variations of chondrocranial elements. The more compact nasal capsule in marsupials is related to a long and strong fixation to the mother’s teat after birth. Within marsupials and placentals, several derived characters distinguish major taxa, probably related to developmental and functional constraints. The reconstructed ancestral anatomy of the cupula nasi anterior supports the hypothesis that the therian ancestor was placental-like and that the marsupial lifestyle is more derived.

Here we document the first stranding record of the pygmy right whale in the Northern Hemisphere—on the coast of The Gambia, Africa (NE Atlantic Ocean, around latitude 13° N)—a location in stark contrast to its current distribution exclusively south of the equator. The original specimen is now missing and untraceable, but a photo found in the files of the Marine Mammal Program, Smithsonian Institution shows sufficient diagnostic features that allow it to be taxonomically identified as the pygmy right whale, Caperea marginata, including: small body size; streamlined overall body shape; generally dark skin coloration; arched rostrum along the lateral margin; triangular and narrow rostrum in dorsal view; lack of head callosities; some fringes on the dorsal surface of the tongue; small and relatively posteriorly positioned dorsal fin; and small and dark-colored flipper. On the whole, a stranding of the pygmy right whale in the Northern Hemisphere, although likely to be a chance event, calls for more detailed studies of how climate change and ocean currents affect the evolution and distribution (re-patterning) of marine mammals and, ultimately, the entire marine ecosystem.

Adult mantis lacewings, neuropteran holometabolan insects of the group Mantispidae, possess anterior walking legs transformed into prey-catching grasping appendages reminiscent of those of praying mantises. While adult mantis lacewings are hence active “wait-and-catch” predators, the larvae of many mantis lacewings have a quite different biology: first-stage larvae seek out female spiders, mount them, and either wait until the spider has produced an egg sac or, in some cases, choose a female already bearing an egg sac. The larva then enters the egg sac and feeds on the eggs. While first stage larvae are highly mobile with comparably long legs and a certain degree of dorso-ventral flattening (“campodeiform”), larval stages two and three are almost immobile, grub-like, and simply remain within the egg sac. Fossils of mantis lacewings are relatively rare, fossils of larval mantis lacewings are even rarer; only a single larva sitting on a juvenile spider has been described from ca. 50 million year old Baltic amber. Here we describe a second occurrence of a larval mantis lacewing from significantly older Burmese amber, about 100 million years old. The specimen is preserved in a position right at the leg of a spider, similar to modern-day larvae that are about to mount their prospective host. The claws of the larva can be seen to grab around the leg of the spider. We discuss how reliable these fossils are as indicators of palaeo-parasitism, and in which aspects the behaviour of mantis lacewing larvae in general indeed represents parasitism. While the specimen appears to be about to board the spider, it may not necessarily represent a parasite in the strict sense. Evaluating the actual ecological role of a fossil heavily depends on comparison to modern forms, and not all modern-day larvae of Mantispidae are parasites. We therefore provide a closer look into the known feeding habits of modern mantis lacewing larvae.

The group Vertebrata is currently placed as a subphylum in the phylum Chordata, together with two other subphyla, Cephalochordata (lancelets) and Urochordata (ascidians). The past three decades, have seen extraordinary advances in zoological taxonomy and the time is now ripe for reassessing whether the subphylum position is truly appropriate for vertebrates, particularly in light of recent advances in molecular phylogeny, comparative genomics, and evolutionary developmental biology. Four lines of current research are discussed here. First, molecular phylogeny has demonstrated that Deuterostomia comprises Ambulacraria (Echinodermata and Hemichordata) and Chordata (Cephalochordata, Urochordata, and Vertebrata), each clade being recognized as a mutually comparable phylum. Second, comparative genomic studies show that vertebrates alone have experienced two rounds of whole-genome duplication, which makes the composition of their gene family unique. Third, comparative gene-expression profiling of vertebrate embryos favors an hourglass pattern of development, the most conserved stage of which is recognized as a phylotypic period characterized by the establishment of a body plan definitively associated with a phylum. This mid-embryonic conservation is supported robustly in vertebrates, but only weakly in chordates. Fourth, certain complex patterns of body plan formation (especially of the head, pharynx, and somites) are recognized throughout the vertebrates, but not in any other animal groups. For these reasons, we suggest that it is more appropriate to recognize vertebrates as an independent phylum, not as a subphylum of the phylum Chordata.

For many years, Paramacrobiotus richtersi was reported to consist of populations with different chromosome numbers and reproductive modes. To clarify the relationships among different populations, the type locality of the species (Clare Island, Ireland) and several Italian localities were sampled. Populations were investigated with an integrated approach, using morphological (LM, CLSM, SEM), morphometric, karyological, and molecular (18S rRNA, cox1 genes) data. Paramacrobiotus richtersi was redescribed and a neotype designed from the Irish bisexual population. Animals of all populations had very similar qualitative and quantitative characters, apart from the absence of males and the presence of triploidy in some of them, whereas some differences were recorded in the egg shell. All populations examined had the same 18S haplotype, while 21 haplotypes were found in the cox1 gene. In four cases, those qualitative characters were correlated with clear molecular (cox1) differences (genetic distance 14.6–21.8%). The integrative approach, which considered the morphological differences in the eggs, the reproductive biology and the wide genetic distances among putative species, led to the description of four new species (Paramacrobiotus arduus sp. n., Paramacrobiotus celsus sp. n., Paramacrobiotus depressus sp. n., Paramacrobiotus spatialis sp. n.) and two Unconfirmed Candidate Species (UCS) within the P. richtersi complex. Paramacrobiotus fairbanksi, the only ascertained parthenogenetic, triploid species, was redescribed and showed a wide distribution (Italy, Spain, Poland, Alaska), while the amphimictic species showed limited distributions. The difference in distribution between apomictic and amphimictic populations can be explained by the difference in the dispersal potentials associated with these two types of reproduction.

Spiral cleavage is a feature of non-ecdysozoan protostomes, in which left-right reversal frequently evolved in gastropod molluscs. In pulmonate gastropods, maternal molecules are responsible for chirality patterning, on which the polarities of visceral and coiling asymmetries depend. In the pond snail, Lymnaea stagnalis (the clade Hygrophila), a frame-shift mutation of one of tandem-duplicated, diaphanous-related formin genes (diaph) resulted in incomplete reversal from dextral to sinistral cleavage. Is this mechanism of chirality regulation common to, or shared with other pulmonates? To answer this question, we examined genes involved in chirality patterning in the land snail, Bradybaena similaris which belongs to the clade Stylommatophora. Both dextral and sinistral siblings develop from progeny of a racemic mutant of B. similaris. Differences in maternal mRNAs between the two strains were searched by transcriptome analyses. We found fifty maternal transcripts that exhibited less expression in early embryos of the mutant strain. The most conspicuous was a homolog of diaph. The diaph gene was duplicated in the stylommatophoran ancestor (diaph-a and diaph-b), as in the case of the ancestor of Lymnaea (Lsdiaph1 and Lsdiaph2). The quantity of maternal diaph-b mRNA was drastically reduced in early embryos of the racemic mutant compared to wild-type, while diaph-a expression was at nearly the same level in both strains. Unlike the case of Lsdiaph2, which is frame-shifted to produce truncated peptides in the mutant of L. stagnalis, however, Bsdiaph-b mRNA in the mutant strain is not frame-shifted and most probably produces normal Diaph-b protein. These results suggest the presence of regulatory mechanisms of gene expression for chirality patterning in pulmonate gastropods, although genomic analyses are required for confirmation. Heterochirality resulting from the loss of polarity control in spiral cleavage does not require mutation of the diaph gene in B. similaris. The determination of left-right polarity instead depends on the expression of this diaph gene, which is duplicated in stylommatophoran Bradybaena, as well as in hygrophilan Lymnaea. Our results provide an avenue to identifying a regulatory mechanism that controls the direction of spiral cleavage in gastropods.

In the context of evolutionary arthopodial transformations, centipede ultimate legs exhibit a plethora of morphological modifications and behavioral adaptations. Many species possess significantly elongated, thickened, or pincer-like ultimate legs. They are frequently sexually dimorphic, indicating a role in courtship and mating. In addition, glandular pores occur more commonly on ultimate legs than on walking legs, indicating a role in secretion, chemical communication, or predator avoidance. In this framework, this study characterizes the evolutionarily transformed ultimate legs in Lithobius forficatus in comparison with regular walking legs. A comparative analysis using macro-photography, SEM, μCT, autofluorescence, backfilling, and 3D-reconstruction illustrates that ultimate legs largely resemble walking legs, but also feature a series of distinctions. Substantial differences are found with regard to aspects of the configuration of specific podomeres, musculature, abundance of epidermal glands, typology and distribution of epidermal sensilla, and architecture of associated nervous system structures. In consideration of morphological and behavioral characteristics, ultimate legs in L. forficatus primarily serve a defensive, but also a sensory function. Moreover, morphologically coherent characteristics in the organization of the ultimate leg versus the antenna-associated neuromere point to constructional constraints in the evolution of primary processing neuropils.

Sea spiders (Pycnogonida) are an abundant faunal element of the Southern Ocean (SO). Several recent phylogeographical studies focused on the remarkably diverse SO pycnogonid fauna, resulting in the identification of new species in previously ill-defined species complexes, insights into their genetic population substructures, and hypotheses on glacial refugia and recolonization events after the last ice age. However, knowledge on the life history of many SO pycnogonids is fragmentary, and early ontogenetic stages often remain poorly documented. This impedes assessing the impact of different developmental pathways on pycnogonid dispersal and distributions and also hinders pycnogonid-wide comparison of developmental features from a phylogenetic-evolutionary angle. Using scanning electron microscopy (SEM) and fluorescent nuclear staining, we studied embryonic stages and postembryonic instars of three SO representatives of the taxon Pallenopsidae (Pallenopsis villosa, P. hodgsoni, P. vanhoeffeni), the development of which being largely unknown. The eggs are large and yolk-rich, and the hatching stage is an advanced lecithotrophic instar that stays attached to the father for additional molts. The first free-living instar is deduced to possess at least three functional walking leg pairs. Despite gross morphological similarities between the congeners, each instar can be reliably assigned to a species based on body size, shape of ocular tubercle and proboscis, structure of the attachment gland processes, and seta patterns on cheliphore and walking legs. We encourage combination of SEM with fluorescent markers in developmental studies on ethanol-preserved and/or long term-stored pycnogonid material, as this reveals internal differentiation processes in addition to external morphology. Using this approach, we describe the first known cases of pallenopsid development with epimorphic tendencies, which stand in contrast to the small hatching larvae in other Pallenopsidae. Evaluation against current phylogenetic hypotheses indicates multiple gains of epimorphic development within Pycnogonida. Further, we suggest that the type of development may impact pycnogonid distribution ranges, since free-living larvae potentially have a better dispersal capability than lecithotrophic attaching instars. Finally, we discuss the bearing of pycnogonid cheliphore development on the evolution of the raptorial first limb pair in Chelicerata and support a multi-articled adult limb as the plesiomorphic state of the chelicerate crown group, arising ontogenetically via postembryonic segmentation of a three-articled embryonic limb.

A major question in stream ecology is how invertebrates cope with flow. In aquatic gastropods, typically, larger and more globular shells with larger apertures are found in lotic (flowing water) versus lentic (stagnant water) habitats. This has been hypothetically linked to a larger foot, and thus attachment area, which has been suggested to be an adaptation against risk of dislodgement by current. Empirical evidence for this is scarce. Furthermore, these previous studies did not discuss the unavoidable increase in drag forces experienced by the snails as a consequence of the increased cross sectional area. Here, using Potamopyrgus antipodarum as a study model, we integrated computational fluid dynamics simulations and a flow tank experiment with living snails to test whether 1) globular shell morphs are an adaptation against dislodgement through lift rather than drag forces, and 2) dislocation velocity is positively linked to foot size, and that the latter can be predicted by shell morphology. The drag forces experienced by the shells were always stronger compared to the lift and lateral forces. Drag and lift forces increased with shell height but not with globularity. Rotating the shells out of the flow direction increased the drag forces, but decreased lift. Our hypothesis that the controversial presence of globular shells in lotic environments could be explained by an adaptation against lift rather than drag forces was rejected. The foot size was only predicted by the size of the shell, not by shell shape or aperture size, showing that the assumed aperture/foot area correlation should be used with caution and cannot be generalized for all aquatic gastropod species. Finally, shell morphology and foot size were not related to the dislodgement speed in our flow tank experiment. We conclude that other traits must play a major role in decreasing dislodgement risk in stream gastropods, e.g., specific behaviours or pedal mucus stickiness. Although we did not find globular shells to be adaptations for reducing dislodgement risk, we cannot rule out that they are still flow-related adaptations. For instance, globular shells are more crush-resistant and therefore perhaps adaptive in terms of diminishing damage caused by tumbling after dislodgement or against lotic crush-type predators.

The pharyngeal arches are a series of bulges found on the lateral surface of the head of vertebrate embryos, and it is within these segments that components of the later anatomy are laid down. In most vertebrates, the post-otic pharyngeal arches will form the branchial apparatus, while in amniotes these segments are believed to generate the larynx. It has been unclear how the development of these segments has been altered with the emergence of the amniotes. In this study, we examined the development of pharyngeal arches in amniotes and show that the post-otic pharyngeal arches in this clade are greatly diminished. We find that the post-otic segments do not undergo myogenesis or skeletogenesis, but are remodelled before these processes occur. We also find that nested DLX expression, which is a feature of all the pharyngeal arches in anamniotes, is associated with the anterior segments but less so with the posterior arches in amniotes. We further show that the posterior arches of the mouse embryo fail to properly delineate, which demonstrates the lack of function of these posterior segments in later development. In amniotes, there has been a loss of the ancestral “branchial” developmental programme that is a general feature of gnathostomes; myogenesis and skeletogenesis This is likely to have facilitated the emergence of the larynx as a new structure not constrained by the segmental organisation of the posterior pharyngeal region.

The free-living marine flatworm Macrostomum lignano is a powerful model organism for use in studying mechanisms of regeneration and stem cell regulation due to its combination of biological and experimental properties, including the availability of transgenesis methods, which is unique among flatworm models. However, due to its relatively recent introduction in research, many aspects of this animal’s biology remain unknown. One such question is the influence of culture temperature on Macrostomum biology. We systematically investigated how different culture temperatures affect development time, reproduction rate, regeneration, heat shock response, and gene knockdown efficiency by RNA interference (RNAi) in M. lignano. We used marker transgenic lines to accurately measure the regeneration endpoint, and to establish the stress response threshold for temperature shock. We found that compared to the culture temperature of 20 °C commonly used for M. lignano, temperatures of 25 °C–30 °C substantially increase the speed of development and regeneration, lead to faster manifestation of RNAi phenotypes, and increase reproduction rate without detectable negative consequences for the animal, while temperatures above 30 °C elicit a heat shock response. We show that altering temperature conditions can be used to reduce the time required to establish M. lignano cultures, perform RNAi experiments, store important lines, and optimize microinjection procedures for transgenesis. These findings will help to optimize the design of experiments in M. lignano, and thus facilitate future research using this model organism.

In the previous paper published in 2017, we described the structure of Hox gene cluster of the ascidian, Halocynthia roretzi, and discussed the scenario for the disintegration of Hox gene clusters during evolution of ascidians. The description about the Hox gene cluster structure still represents the latest information, hence it has been left unchanged. In contrast, some points in Discussion, the description on the phylogenetic relationships among tunicates and the theoretical scenario for the disintegration of Hox gene cluster during evolution of ascidians, should be changed because the phylogenetic relationships among tunicates have recently been updated. The above mentioned points were made in accordance with the phylogenetic tree for tunicates based on the mitochondrial DNA sequences, which was the latest at the time of publication. In 2018, however, Kocot et al. and Delsuc et al. proposed new phylogenetic trees for tunicates based on a large number of nuclear gene sequences. The trees obtained by the two groups are essentially the same and different from the previous one in the phylogenetic positions of Appendicularia and Thaliacea, which leads to a change in the order of the emergence of ascidians and the Hox gene cluster disintegration during evolution of ascidians or tunicates. We add here a note to update the previous description on the phylogenetic relationships among tunicates and the theoretical scenario, including one Figure, so as to coincide with the new phylogenetic relationships among tunicates based on the nuclear gene sequences. The previous summarized conclusion remains unchanged: we suggest that the Hox gene cluster of the ancestral ascidian experienced extensive genome shuffling during the course of evolution to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene organization on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to all ascidians and later lineage-specific events.

In the darkness of the ocean, an impressive number of taxa have evolved the capability to emit light. Many mesopelagic organisms emit a dim ventral glow that matches with the residual environmental light in order to camouflage themselves (counterillumination function). Sharks use their luminescence mainly for this purpose. Specific lateral marks have been observed in Etmopteridae sharks (one of the two known luminous shark families) suggesting an inter/intraspecific recognition. Conversely, dorsal luminescence patterns are rare within these deep-sea organisms. Here we report evidence that Etmopterus spinax, Etmopterus molleri and Etmopterus splendidus have dorsal luminescence patterns. These dorsal patterns consist of specific lines of luminous organs, called photophores, on the rostrum, dorsal area and at periphery of the spine. This dorsal light seems to be in contrast with the counterilluminating role of ventral photophores. However, skin photophores surrounding the defensive dorsal spines show a precise pattern supporting an aposematism function for this bioluminescence. Using in situ imaging, morphological and histological analysis, we reconstructed the dorsal light emission pattern on these species, with an emphasis on the photogenic skin associated with the spine. Analyses of video footage validated, for the first time, the defensive function of the dorsal spines. Finally, we did not find evidence that Etmopteridae possess venomous spine-associated glands, present in Squalidae and Heterondontidae, via MRI and CT scans. This work highlights for the first time a species-specific luminous dorsal pattern in three deep-sea lanternsharks. We suggest an aposematic use of luminescence to reveal the presence of the dorsal spines. Despite the absence of venom apparatus, the defensive use of spines is documented for the first time in situ by video recordings.

Day–night behavioral variation is observed in most organisms, and is generally controlled by circadian clocks and/or synchronization to environmental cues. Hydra species, which are freshwater cnidarians, are thought to lack the core clock genes that form transcription–translation feedback loops in clock systems. In this study, we examined whether hydras exhibit diel rhythms in terms of behavior and gene expression levels without typical clock genes. We found that the total behavior of hydras was elevated during the day and decreased at night under a 12-h light–dark cycle. Polyp contraction frequency, one component of behavior, exhibited a clear diel rhythm. However, neither total behavior nor polyp contraction frequency showed rhythmic changes under constant light and constant dark conditions. To identify the genes underlying diel behavior, we performed genome-wide transcriptome analysis of hydras under light–dark cycles. Using three different analytic algorithms, we found that 380 genes showed robust diel oscillations in expression. Some of these genes shared common features with diel cycle genes of other cnidarian species with endogenous clock systems. Hydras show diel behavioral rhythms under light–dark cycles despite the absence of canonical core clock genes. Given the functions of the genes showing diel oscillations in hydras and the similarities of those genes with the diel cycle genes of other cnidarian species with circadian clocks, it is possible that diel cycle genes play an important role across cnidarian species regardless of the presence or absence of core clock genes under light–dark cycles.

The dentition in extant holocephalans (Chondrichthyes) comprises three pairs of continuously growing dental plates, rather than the separate teeth characterizing elasmobranchs. We investigated how different types of dentine in these plates, including hypermineralized dentine, are arranged, and how this is renewed aborally, in adult and juvenile dentitions of Harriotta raleighana (Rhinochimeridae). Individual plates were analysed using x-ray computed tomography (μCT), scanning electron microscopy (SEM) in back scattered mode with energy dispersive X-ray (EDX) analysis, and optical microscopy on hard tissue sections. Harriotta dental plates are made entirely of dentine tissue, mostly as trabecular dentine, bone itself being absent. Hypermineralized dentine forms in restricted ovoid and tritor spaces within trabecular dentine, inside a shell of outer and inner dentine layers. Trabecular dentine is ubiquitous but changes to sclerotic osteodentine near the oral surface by increasing density, remaining less mineralized than the hypermineralized dentine. All structures are renewed aborally, within a vascular dental pulp, a tissue suggested to be a source of stem cells for tissue renewal. Ca density profiles and concentrations of Mg, P, and Ca ions reveal extreme differences in the level and type of mineralization. Early mineralization in ovoids and tritors has very high levels of Mg, then a sudden increase in mineralization to a high total mineral content, whereas there is gradual change in trabecular dentine, remaining at a low level. Hypermineralized dentine fills the prepatterned ovoid, rod and tritor spaces, early at the aboral surface within the trabecular dentine. Deposition of the hypermineralized dentine (HD, proposed as new specific name, whitlockin replacing pleromin) is from surfaces that are lined with large specialized odontoblasts, (whitloblasts, instead of pleromoblasts) within cell body spaces connecting with extensive, ramifying tubules. Early mineralization occurs amongst this maze of tubules that penetrate far into the dentine, expanding into a mass of saccules and membranous bodies, dominating in the absence of other organic matrix. This early stage has hydroxyapatite, also significantly rich in Mg, initiated as a poorly crystalline phase. In the hypermineralized dentine, formation occurs as clusters of variably shaped crystals, arising from a sudden phase transition. In the hypermineralized dentine, high MgO + CaO + P2O5 suggests that almost pure Mg containing tricalciumphosphate (MgTCP: (ß-Ca3(PO4)2) (whitlockite) is present, with little or no hydroxyapatite. Serial replacement of tritors and ovoids is suggested to occur within the dental plate, probably representing a relic of patterning, as classically found in elasmobranch dentitions.

Branched structures are found in many natural settings, and the molecular and cellular mechanisms underlying their formation in animal development have extensively studied in recent years. Despite their importance and the accumulated knowledge from studies on several organs of Drosophila and mammals, much remains unknown about branching mechanisms in other animal species. We chose to study the jellyfish species Cladonema pacificum. Unlike many other jellyfish, this species has branched medusa tentacles, and its basal phylogenetic position in animal evolution makes it an ideal organism for studying and understanding branching morphogenesis more broadly. Branched tentacles are unique compared to other well-studied branched structures in that they have two functionally distinct identities: one with adhesive organs for attaching to a substratum, and another with nematocyst clusters for capturing prey. We began our analyses on C. pacificum tentacles by observing their branching during growth. We found that tentacle branches form through repeated addition of new branches to the proximal region of the main tentacle while it is elongating. At the site of branch bud formation, we observed apical thickening of the epidermal epithelial layer, possibly caused by extension of the epithelial cells along the apico-basal axis. Interestingly, tentacle branch formation required receptor tyrosine kinase signaling, which is an essential factor for branching morphogenesis in Drosophila and mammals. We also found that new branches form adhesive organs first, and then are transformed into branches with nematocyst clusters as they develop. These results highlight unique features in branch generation in C. pacificum medusa tentacles and illuminate conserved and fundamental mechanisms by which branched structures are created across a variety of animal species.

End-Cretaceous niche-filling by benthic Mesozoic survivors resulted in a prominent increase of durophagous fish families, resulting in the appearance of the earliest representatives of several extant fish lineages, including the pelagic durophagous stingrays, a monophyletic clade of myliobatiform batoids that is characterized by a derived swimming mode and feeding habits. Although the earliest members appeared in the Late Cretaceous, most of the crown genera date back to the Eocene. In this study, we re-examine the anatomy of the Eocene eagle ray Promyliobatis gazolai (de Zigno), represented by two nearly complete and articulated specimens from the world-famous Ypresian Konservat-Lagerstätte of Bolca, in detail. This taxon exhibits a mosaic of plesiomorphic and derived characters (e.g. tail sting displaced posteriorly on the tail, at about 50–60% of tail length; pectoral fins joining in front of the head; anterior and posterior pectoral fin margins nearly straight; compagibus laminam absent; single, unfragmented mesopterygium) that clearly define a new body plan within the pelagic durophagous stingrays. The significant morphological differences between Promyliobatis and extant representatives of Myliobatidae, Aetobatidae, Rhinopteridae, and Mobulidae, support its placement as separate stem group member. The phylogenetic placement of Promyliobatis, based on skeletal and dental characters, strongly supports its basal position within pelagic stingrays. However, its position within the Myliobatiformes becomes unstable when stingray taxa known by fossil teeth only are included. A comparative analysis of the skeletal and tooth morphologies, as well as of the evolutionary trends of pelagic stingrays is also discussed.

Tardigrades (water bears) are microscopic invertebrates of which the anatomy has been well studied using traditional techniques, but a comprehensive three-dimensional reconstruction has never been performed. In order to close this gap, we employed X-ray computed tomography (CT), a technique that is becoming increasingly popular in zoology for producing high-resolution, three-dimensional (3D) scans of whole specimens. While CT has long been used to scan larger samples, its use in some microscopic animals can be problematic, as they are often too small for conventional CT yet too large for high-resolution, optics-based soft X-ray microscopy. This size gap continues to be narrowed with advancements in technology, with high-resolution imaging now being possible using both large synchrotron devices and, more recently, laboratory-based instruments. Here we use a recently developed prototype lab-based nano-computed tomography device to image a 152 μm-long tardigrade at high resolution (200–270 nm pixel size). The resulting dataset allowed us to visualize the anatomy of the tardigrade in 3D and analyze the spatial relationships of the internal structures. Segmentation of the major structures of the body enabled the direct measurement of their respective volumes. Furthermore, we segmented every storage cell individually and quantified their volume distribution. We compare our measurements to those from published studies in which other techniques were used. The data presented herein demonstrate the utility of CT imaging as a powerful supplementary tool for studies of tardigrade anatomy, especially for quantitative volume measurements. This nanoCT study represents the smallest complete animal ever imaged using CT, and offers new 3D insights into the spatial relationships of the internal organs of water bears.

The crustacean class Branchiopoda includes fairy shrimps, clam shrimps, tadpole shrimps, and water fleas. Branchiopods, which are well known for their great variety of reproductive strategies, date back to the Cambrian and extant taxa can be mainly found in freshwater habitats, also including ephemeral ponds. Mitochondrial genomes of the notostracan taxa Lepidurus apus lubbocki (Italy), L. arcticus (Iceland) and Triops cancriformis (an Italian and a Spanish population) are here characterized for the first time and analyzed together with available branchiopod mitogenomes. Overall, branchiopod mitogenomes share the basic structure congruent with the ancestral Pancrustacea model. On the other hand, rearrangements involving tRNAs and the control region are observed among analyzed taxa. Remarkably, an unassigned region in the L. apus lubbocki mitogenome showed a chimeric structure, likely resulting from a non-homologous recombination event between the two flanking trnC and trnY genes. Notably, Anostraca and Onychocaudata mitogenomes showed increased GC content compared to both Notostraca and the common ancestor, and a significantly higher substitution rate, which does not correlate with selective pressures, as suggested by dN/dS values. Branchiopod mitogenomes appear rather well-conserved, although gene rearrangements have occurred. For the first time, it is reported a putative non-homologous recombination event involving a mitogenome, which produced a pseudogenic tRNA sequence. In addition, in line with data in the literature, we explain the higher substitution rate of Anostraca and Onychocaudata with the inferred GC substitution bias that occurred during their evolution.

Diverse insects are intimately associated with microbial symbionts, which play a variety of biological roles in their adaptation to and survival in the natural environment. Such insects often possess specialized organs for hosting the microbial symbionts. What developmental processes and mechanisms underlie the formation of the host organs for microbial symbiosis is of fundamental biological interest but poorly understood. Here we investigate the morphogenesis of the midgut symbiotic organ and the process of symbiont colonization therein during the developmental course of the stinkbug Plautia stali. Upon hatching, the midgut is a simple and smooth tube. Subsequently, symbiont colonization to the posterior midgut occurs, and thickening and folding of the midgut epithelium proceed during the first instar period. By the second instar, rudimentary crypts have formed, and their inner cavities are colonized by the symbiotic bacteria. From the second instar to the fourth instar, while the alimentary tract grows and the posterior midgut is established as the symbiotic organ with numerous crypts, the anterior midgut and the posterior midgut are structurally and functionally isolated by a strong constriction in the middle. By the early fifth instar, the midgut symbiotic organ attains the maximal length, but toward the mid fifth instar, the basal region of each crypt starts to constrict and narrow, which deforms the midgut symbiotic organ as a whole into a shorter, thicker and twisted shape. By the late fifth instar to adulthood, the crypts are constricted off, by which the symbiotic bacteria are confined in the crypt cavities and isolated from the midgut main tract, and concurrently, the strong midgut constriction in the middle becomes loose and open, by which the food flow from the anterior midgut to the posterior midgut recovers. This study provides the most detailed and comprehensive descriptions ever reported on the morphogenesis of the symbiotic organ and the process of symbiont colonization in an obligatory insect-bacterium gut symbiotic system. Considering that P. stali is recently emerging as a useful model system for experimentally studying the intimate insect-microbe gut symbiosis, the knowledge obtained in this study establishes the foundation for the further development of this research field.

The rock dove (or common pigeon), Columba livia, is an important model organism in biological studies, including research focusing on head muscle anatomy, feeding kinematics, and cranial kinesis. However, no integrated computer-based biomechanical model of the pigeon head has yet been attempted. As an initial step towards achieving this goal, we present the first three-dimensional digital dissection of the pigeon head based on a contrast-enhanced computed tomographic dataset achieved using iodine potassium iodide as a staining agent. Our datasets enable us to visualize the skeletal and muscular anatomy, brain and cranial nerves, and major sense organs of the pigeon, including very small and fragile features, as well as maintaining the three-dimensional topology of anatomical structures. This work updates and supplements earlier anatomical work on this widely used laboratory organism. We resolve several key points of disagreement arising from previous descriptions of pigeon anatomy, including the precise arrangement of the external adductor muscles and their relationship to the posterior adductor. Examination of the eye muscles highlights differences between avian taxa and shows that pigeon eye muscles are more similar to those of a tinamou than they are to those of a house sparrow. Furthermore, we present our three-dimensional data as publicly accessible files for further research and education purposes. Digital dissection permits exceptional visualisation and will be a valuable resource for further investigations into the head anatomy of other bird species, as well as efforts to reconstruct soft tissues in fossil archosaurs.

Radiodonta, large Palaeozoic nektonic predators, occupy a pivotal evolutionary position as stem-euarthropods and filled important ecological niches in early animal ecosystems. Analyses of the anatomy and phylogenetic affinity of these large nektonic animals have revealed the origins of the euarthropod compound eye and biramous limb, and interpretations of their diverse feeding styles have placed various radiodont taxa as primary consumers and apex predators. Critical to our understanding of both radiodont evolution and ecology are the paired frontal appendages; however, the vast differences in frontal appendage morphology between and within different radiodont families have made it difficult to identify the relative timings of character acquisitions for this body part. Here we describe a new genus of hurdiid, Ursulinacaris, from the middle Cambrian (Miaolingian, Wuliuan) Mount Cap Formation (Northwest Territories, Canada) and Jangle Limestone (Nevada, USA). Ursulinacaris has the same organisation as other hurdiid frontal appendages, with elongate endites on the first five podomeres in the distal articulated region and auxiliary spines on the distal margin of endites only. Unlike all other hurdiid genera, which possess a single row of elongated and blade-like ventral endites, this taxon uniquely bears paired slender endites. The blade-like endite morphology is shown to be a hurdiid autapomorphy. Two other frontal appendage characters known only in hurdiids, namely auxiliary spines on the distal margin of endites only, and elongate endites on the first five podomeres in the distal articulated region only, predate this innovation.

Catfish (Siluriformes) are characterized by unique morphologies, including enlarged jaws with movable barbels and taste buds covering the entire body surface. Evolution of these characteristics was a crucial step in their adaptive radiation to freshwater environments. However, the developmental processes of the catfish craniofacial region and taste buds remain to be elucidated; moreover, little is known about the molecular mechanisms underlying the morphogenesis of these structures. In Amur catfish (Silurus asotus), three pairs of barbel primordia are formed by 2 days post-fertilization (dpf). Innervation of the peripheral nerves and formation of muscle precursors are also established during early development. Taste buds from the oral region to the body trunk are formed by 4 dpf. We then isolated catfish cognates Shh (SaShh) and Fgf8 (SaFgf8), which are expressed in maxillary barbel primordium at 1–2 dpf. Further, SHH signal inhibition induces reduction of mandibular barbels with abnormal morphology of skeletal elements, whereas it causes no apparent abnormality in the trigeminal and facial nerve morphology. We also found that mandibular barbel lengths and number of taste buds are reduced by FGF inhibition, as seen in SHH signal inhibition. However, unlike with SHH inhibition, the abnormal morphology of the trigeminal and facial nerves was observed in FGF signal-inhibited embryos. The developmental processes of Amur catfish are consistent with those reported for other catfish species. Thus, developmental aspects of craniofacial structures and taste buds may be conserved in Siluriformes. Our findings also suggest that SHH signaling plays a crucial role in the formation of barbels and taste buds, without affecting nerve projection, while FGF signaling is required for the development of barbels, taste buds, and branchial nerves. Thus, SHH and FGF signaling plays key roles in the ontogenesis and evolution of some catfish-specific characteristics.

Calmanostraca is a group of branchiopod eucrustaceans, with Triops cancriformis and Lepidurus apus as most prominent representatives. Both are regularly addressed with the inaccurate tag “living fossil”, suggesting that the morphology has remained stable for several millions of years. Yet, T. cancriformis and L. apus represent only a fraction of the morphological diversity occurring in Calmanostraca, comprising the two groups Notostraca and Kazacharthra. Notostracans, commonly called tadpole shrimps, comprise the two groups Lepidurus and Triops with their elongated and rather narrow (in dorsal view) head shields. Kazacharthrans are exclusively fossil calmanostracans with broad and rather short shields, known from the Jurassic and Triassic period. One formation where fossil calmanostracans have been found is the Yixian Formation of northeastern China (Lower Cretaceous, 125–121 million years). It is part of the Jehol Group, an ecosystem known for its exceptionally well-preserved fossils, including vertebrates and plants, but also diverse arthropods. Two calmanostracan species have to date been described from the Yixian Formation, Jeholops hongi and Chenops yixianensis. We describe here a new calmanostracan crustacean from the Yixian Formation, Notostraca oleseni, and additionally a simple tool using a morphospace analysis to delineate different species. Measurements characterising the shield and trunk proportions of different calmanostracan species were performed, data were size-corrected, and used for this morphospace analysis to compare the different morphologies. As sclerotised body parts are more likely to be preserved in fossils than soft tissue, shields and parts of the trunk are in many cases the only morphological structures available for study. Therefore, the present analysis represents a simple tool for distinguishing between different species, as well as allowing the inclusion of specimens that are only preserved fragmentarily. Additionally, it provides a tool to demarcate the kazacharthran-like specimen described, but not formally named, by Wagner et al. (Paleontol Res. 22:57–63, 2018). Hence, we amended the description and name the species Calmanostraca hassbergella. Our results indicate a large diversity in shield and trunk morphology in calmanostracans, in contrast to their often claimed highly conserved and uniform morphology. Especially extinct forms such as Notostraca oleseni add up to this result and point to the species richness and morphological diversity within Calmanostraca.

Sea cucumbers (a class of echinoderms) exhibit a high capacity for regeneration, such that, following ejection of inner organs in a process called evisceration, the lost organs regenerate. There are two ways by which evisceration occurs in sea cucmber species: from the mouth (anterior) or the anus (posterior). Intriguingly, regenerating tissues are formed at both the anterior and posterior regions and extend toward the opposite ends, and merge to form a complete digestive tract. From the posterior side, the digestive tube regenerates extending a continuous tube from the cloaca, which remains at evisceration. In posteriorly-eviscerating species, the esophagus remains in the body, and a new tube regenerates continuously from it. However, in anterior-eviscerating species, no tubular tissue remains in the anterior region, raising the question of how the new digestive tube forms in the anterior regenerate. We addressed this question by detailed histological observations of the regenerating anterior digestive tract in a small sea cucumber, Eupentacta quinquesemita (“ishiko” in Japanese) after induced-evisceration. We found that an initial rudiment consisting of mesenchymal cells is formed along the edge of the anterior mesentery from the anterior end, and then, among the mesenchymal cells, multiple clusters of epithelial-like cells appears simultaneously and repeatedly in the extending region by mesenchymal–epithelial transition (MET) as visulalized using toluidine blue staining. Subsequently, multiple cavities were formed surrounded with these epithelial cells, and appeared to coalesce with each other to form into multiple lumens, and to eventually become a single tube. This anterior tube then fused to the tube regenerated from the posterior rudiment. Thus, we elucidated the process of regeneration of the anterior portion of the gut in an anteriorly eviscerating species, and suggest the involvement of MET and fusion of cavities/lumens in regeneration of the digestive tube.

Please note that there are two errors present in the tables of the published article [1].

Gonadotropin releasing hormone (GnRH) is a hypophysiotropic hormone that is generally thought to be important for reproduction. This hormone is produced by hypothalamic GnRH neurons and stimulates the secretion of gonadotropins. On the other hand, vertebrates also have non-hypophysiotropic GnRH peptides, which are produced by extrahypothalamic GnRH neurons. They are mainly located in the terminal nerve, midbrain tegmentum, trigeminal nerve, and spinal cord (sympathetic preganglionic nerves). In vertebrates, there are typically three gnrh paralogues (gnrh1, gnrh2, gnrh3). GnRH-expression in the non-hypophysiotropic neurons (gnrh1 or gnrh3 in the terminal nerve and the trigeminal nerve, gnrh2 in the midbrain tegmentum) occurs from the early developmental stages. Recent studies have suggested that non-hypophysiotropic GnRH neurons play various functional roles. Here, we summarize their anatomical/physiological properties and discuss their possible functions, focusing on studies in vertebrates. GnRH neurons in the terminal nerve show different spontaneous firing properties during the developmental stages. These neurons in adulthood show regular pacemaker firing, and it has been suggested that these neurons show neuromodulatory function related to the regulation of behavioral motivation, etc. In addition to their recognized role in neuromodulation in adult, in juvenile fish, these neurons, which show more frequent burst firing than in adults, are suggested to have novel functions. GnRH neurons in the midbrain tegmentum show regular pacemaker firing similar to that of the adult terminal nerve and are suggested to be involved in modulations of feeding (teleosts) or nutrition-related sexual behaviors (musk shrew). GnRH neurons in the trigeminal nerve are suggested to be involved in nociception and chemosensory avoidance, although the literature on their electrophysiological properties is limited. Sympathetic preganglionic cells in the spinal cord were first reported as peptidergic modulatory neurons releasing GnRH with a putative function in coordinating interaction between vasomotor and exocrine outflow in the sympathetic nervous system. The functional role of non-hypophysiotropic GnRH neurons may thus be in the global modulation of neural circuits in a manner dependent on internal conditions or the external environment.

Paedomorphosis describes the retention of larval characters in adult stages and is widespread amongst salamanders. Salamandrid newts exhibit facultative paedomorphosis, where paedomorphic and metamorphic adult forms coexist in the same population. Previous studies have shown that prey capture kinematics do not differ between paedomorphic and metamorphosed ambystomatid salamanders, despite diverging morphology and prey capture performance. It remained unclear, however, whether the stereotypy of prey capture kinematics across morphotypes is restricted to ambystomatids, or can be found in other salamander groups too. Here, we performed biplanar high-speed-recordings of the prey capture behavior in paedomorphic and metamorphic salamandrid newts and only found minor kinematic differences across morphotypes, suggesting that stereotypy across morphotypes is a more general feature within salamanders. We then compared anatomy of skull and hyobranchial skeleton, along with the physiological cross sectional area (PCSA) of the rectus cervicis muscle, the main muscle empowering suction feeding. Besides the overall morphological differences of the feeding apparatus, the PCSA of the rectus cervicis also differs significantly between morphotypes, being twice as large in paedomorphs. Accordingly, paedomorphs can exert more powerful suction strikes, which in turn may be one of the key factors why paedomorphs are more efficient in capturing elusive prey compared to metamorphs.

CatSper is a sperm-specific calcium ion (Ca2+) channel, which regulates sperm flagellar beating by tuning cytoplasmic Ca2+ concentrations. Although this Ca2+ channel is essential for mammalian fertilization, recent bioinformatics analyses have revealed that genes encoding CatSper are heterogeneously distributed throughout the eukaryotes, including vertebrates. As this channel is activated by cytoplasmic alkalization in mammals and sea urchins, it has been proposed that the sperm-specific Na+/H+ exchanger (sNHE, a product of the SLC9C gene family) positively regulates its activity. In mouse, sNHE is functionally coupled to soluble adenylyl cyclase (sAC). CatSper, sNHE, and sAC have thus been considered functionally interconnected in the control of sperm motility, at least in mouse and sea urchin. We carried out a comparative genomic analysis to explore phylogenetic relationships among CatSper, sNHE and sAC in eukaryotes. We found that sNHE occurs only in Metazoa, although sAC occurs widely across eukaryotes. In animals, we found correlated and restricted distribution patterns of the three proteins, suggesting coevolution among them in the Metazoa. Namely, nearly all species in which CatSper is conserved also preserve sNHE and sAC. In contrast, in species without sAC, neither CatSper nor sNHE is conserved. On the other hand, the distribution of another testis-specific NHE (NHA, a product of the SLC9B gene family) does not show any apparent association with that of CatSper. Our results suggest that CatSper, sNHE and sAC form prototypical machinery that functions in regulating sperm flagellar beating in Metazoa. In non-metazoan species, CatSper may be regulated by other H+ transporters, or its activity might be independent of cytoplasmic pH.

Arachnids possess highly specialized and unorthodox sense organs, such as the unique pectines of Scorpiones and the malleoli of Solifugae. While the external morphology, numbers, and shapes of sensory organs are widely used in taxonomic studies, little is known about the internal anatomy of these organs and their associated processing neuropils in the central nervous system. Camel spiders (Solifugae) possess pedipalps and first walking legs heavily endowed with sensory structures, as well as conspicuous malleoli located ventrally on the proximal fourth walking legs. Malleoli are fan-shaped organs that contain tens of thousands of presumptive chemoreceptor neurons, but mechanoreceptive structures are absent. Here, we examine the organization of the synganglion based on microCT analysis, 3D reconstruction of serial paraffin sections, and backfill preparations to trace the malleolar pathway. The projection area of malleolar afferents is intriguingly located in the most anterior ventral nerve cord, located in between the pedipalpal neuromere hemispheres. However, malleolar axon bundles are separated by a thin soma layer that points to an anteriad projection of the fourth walking leg neuromere. A conspicuous projection neuron tract that may receive additional input from pedipalpal sensory organs connects the malleolar neuropil with the mushroom bodies in the protocerebrum. Arthropod chemosensory appendages or organs and primary processing neuropils are typically located in the same segment, which also holds true in Solifugae, although the malleolar neuropil is partially shifted towards the pedipalpal neuromere. A comparison of the malleoli in Solifugae and the pectines in Scorpiones, and of their primary processing neuropils, reveals certain similarities, while striking differences are also evident. Similarities include the ventral arrangement of peg-shaped sensory structures on the respective segmental appendage, exposing dense arrays of chemoreceptive sensilla, and projections to a primary processing neuropil with glomerular subdivision. Differences are, e.g., the lack of mechanoreceptive afferents and an associated processing neuropil.

In cephalochordates (amphioxus), the notochord runs along the dorsal to the anterior tip of the body. In contrast, the vertebrate head is formed anterior to the notochord, as a result of head organizer formation in anterior mesoderm during early development. A key gene for the vertebrate head organizer, goosecoid (gsc), is broadly expressed in the dorsal mesoderm of amphioxus gastrula. Amphioxus gsc expression subsequently becomes restricted to the posterior notochord from the early neurula. This has prompted the hypothesis that a change in expression patterns of gsc led to development of the vertebrate head during chordate evolution. However, molecular mechanisms of head organizer evolution involving gsc have never been elucidated. To address this question, we compared cis-regulatory modules of vertebrate organizer genes between amphioxus, Branchiostoma japonicum, and frogs, Xenopus laevis and Xenopus tropicalis. Here we show conservation and diversification of gene regulatory mechanisms through cis-regulatory modules for gsc, lim1/lhx1, and chordin in Branchiostoma and Xenopus. Reporter analysis using Xenopus embryos demonstrates that activation of gsc by Nodal/FoxH1 signal through the 5′ upstream region, that of lim1 by Nodal/FoxH1 signal through the first intron, and that of chordin by Lim1 through the second intron, are conserved between amphioxus and Xenopus. However, activation of gsc by Lim1 and Otx through the 5′ upstream region in Xenopus are not conserved in amphioxus. Furthermore, the 5′ region of amphioxus gsc recapitulated the amphioxus-like posterior mesoderm expression of the reporter gene in transgenic Xenopus embryos. On the basis of this study, we propose a model, in which the gsc gene acquired the cis-regulatory module bound with Lim1 and Otx at its 5′ upstream region to be activated persistently in anterior mesoderm, in the vertebrate lineage. Because Gsc globally represses trunk (notochord) genes in the vertebrate head organizer, this cooption of gsc in vertebrates appears to have resulted in inhibition of trunk genes and acquisition of the head organizer and its derivative prechordal plate.

Bryozoa is a large phylum of colonial aquatic suspension feeders. The boring ctenostome Hypophorella expansa is unique and inhabits parchment-like polychaete tubes. Morphological studies date back to the nineteenth century, but distinct adaptations to this specific habitat have not been properly analysed, which prompted us to reexamine the morphology of this recently encountered species. The colony of H. expansa is composed of elongated stolonal kenozooids with a distal capsule-like expansion. A median transversal muscle is present in the latter, and one autozooid is laterally attached to the capsule. Unique stolonal wrinkles are embedded in the thin parts of the stolons. Single autozooids are attached in an alternating right–left succession on subsequent stolons. Polypide morphology including digestive tract, muscular system and most parts of the nervous system are similar to other ctenostomes. The most obvious apomorphic features of Hypophorella are space balloons and the gnawing apparatus. The former are two fronto-lateral spherical structures on autozooids, which provide space inside the tube. The latter perforates layers of the polychaete tube wall and consists of two rows of cuticular teeth that, together with the entire vestibular wall, are introvertable during the protrusion-retraction process. The apertural muscles are in association with this gnawing apparatus heavily modified and show bilateral symmetry. Adaptations to the unique lifestyle of this species are thus evident in stolonal wrinkles, autozooidal space balloons and the gnawing apparatus. The growth pattern of the colony of H. expansa may aid in rapid colonization of the polychaete tube layers.

Biological diversity is a hot topic in current research, especially its observed decrease in modern times. Investigations of past ecosystems offer additional insights to help better understand the processes underlying biodiversity. The Cretaceous period is of special interest in this context, especially with respect to arthropods. During that period, representatives of many modern lineages appeared for the first time, while representatives of more ancient groups also co-occurred. At the same time, side branches of radiating groups with ‘experimental morphologies’ emerged that seemed to go extinct shortly afterwards. However, larval forms, with their morphological diversity, are largely neglected in such studies, but may provide important insights into morphological and ecological diversity and its changes in the past. We present here a new fossil insectan larva, a larval lacewing, in Cretaceous amber, exhibiting a rather unusual, ‘experimental’ morphology. The specimen possesses extremely large (in relation to body size) mandibulo-maxillary piercing stylets. Additionally, the labial palps are very long and are subdivided into numerous elements, overall appearing antenniform. In other aspects, the larva resembles many other neuropteran-type larvae. We provide a comparison that includes quantitative aspects of different types of neuropteran larvae to emphasise the exceptionality of the new larva, and discuss its possible relationships to known lineages of Neuroptera; possible interpretations are closer relationships to Dilaridae or Osmylidae. In any case, several of the observed characters must have evolved convergently. With this new find, we expand the known morphological diversity of neuropterans in the Cretaceous fauna.

Teleost paired fins are composed of two endoskeletal domains, proximal and distal radials, and an exoskeletal domain, the fin ray. The zebrafish pectoral fin displays elaborately patterned radials along the anteroposterior (AP) axis. Radials are considered homologous to tetrapod limb skeletons, and their patterning mechanisms in embryonic development are similar to those of limb development. Nevertheless, the pattern along the AP axis in fin rays has not been well described in the zebrafish pectoral fin, although several recent reports have revealed that fin ray development shares some cellular and genetic properties with fin/limb endoskeleton development. Thus, fin ray morphogenesis may involve developmental mechanisms for AP patterning in the fin/limb endoskeleton, and may have a specific pattern along the AP axis. We conducted detailed morphological observations on fin rays and their connection to distal radials by comparing intra- and inter-strain zebrafish specimens. Although the number of fin rays varied, pectoral fin rays could be categorized into three domains along the AP axis, according to the connection between the fin rays and distal radials; additionally, the number of fin rays varied in the posterior part of the three domains. This result was confirmed by observation of the morphogenesis process of fin rays and distal radials, which showed altered localization of distal radials in the middle domain. We also evaluated the expression pattern of lhx genes, which have AP patterning activity in limb development, in fin rays and during distal radial development and found these genes to be expressed during morphogenesis in both fin rays and distal radials. The fin ray and its connection to the endoskeleton are patterned along the AP axis, and the pattern along the AP axis in the fin ray and the radial connection is constructed by the developmental mechanism related to AP patterning in the limb/fin bud. Our results indicate the possibility that the developmental mechanisms of fin rays and their connection are comparable to those of the distal element of the limb skeleton.