The sea spider Pycnogonum litorale overturns the paradigm of the absence of axial regeneration in molting animals.

Regenerative abilities and their evolution in the different animal lineages have fascinated generations of biologists. While some taxa are capable of restoring entire individuals from small body fragments, others can regrow only specific structures or lack structural regeneration completely. In contrast to many other protostomes, including the segmented annelids, molting animals (Ecdysozoa) are commonly considered incapable of primary body axis regeneration, which has been hypothesized to be linked to the evolution of their protective cuticular exoskeleton. This holds also for the extraordinarily diverse, segmented arthropods. Contradicting this long-standing paradigm, we here show that immatures of the sea spider Pycnogonum litorale reestablish the posterior body pole after transverse amputation and can regrow almost complete segments and the terminal body region, including the hindgut, anus, and musculature. Depending on the amputation level, normal phenotypes or hypomeric six-legged forms develop. Remarkably, also the hypomeric animals regain reproductive functionality by ectopic formation of gonoducts and gonopores. The discovery of such complex regenerative patterns in an extant arthropod challenges the hitherto widely assumed evolutionary loss of axial regeneration during ecdysozoan evolution. Rather, the branching of sea spiders at the base of Chelicerata and their likely ancestral anamorphic development suggests that the arthropod stem species may have featured similar regenerative capabilities. Accordingly, our results provide an incentive for renewed comparative regeneration studies across ecdysozoans, with the aim to resolve whether this trait was potentially even inherited from the protostome ancestor.

Gradients of Orientation, Composition, and Hydration of Proteins for Efficient Light Collection by the Cornea of the Horseshoe Crab.

The lateral eyes of the horseshoe crab, Limulus polyphemus, are the largest compound eyes within recent Arthropoda. The cornea of these eyes contains hundreds of inward projecting elongated cuticular cones and concentrate light onto proximal photoreceptor cells. Although this visual system has been extensively studied before, the precise mechanism allowing vision has remained controversial. Correlating high-resolution quantitative refractive index (RI) mapping and structural analysis, it is demonstrated how gradients of RI in the cornea stem from structural and compositional gradients in the cornea. In particular, these RI variations result from the chitin-protein fibers architecture, heterogeneity in protein composition, and bromine doping, as well as spatial variation in water content resulting from matrix cross-linking on the one hand and cuticle porosity on the other hand. Combining the realistic cornea structure and measured RI gradients with full-wave optical modeling and ray tracing, it is revealed that the light collection mechanism switches from refraction-based graded index (GRIN) optics at normal light incidence to combined GRIN and total internal reflection mechanism at high incident angles. The optical properties of the cornea are governed by different mechanisms at different hierarchical levels, demonstrating the remarkable versatility of arthropod cuticle.

Screwed up: Spirality of segments and other iterated structures suggest an underlying principle of seriality in bilaterians.

This review deals with helicomery, that is, the specific malformation of a spiral arrangement of segments and other serial structures. Helicomery was first described in annelid and arthropod body segments. However, corresponding patterns occur in arthropod appendages and other bilaterians with serially arranged body parts, such as tapeworms, nematodes, vertebrates, and probably chitons. The specifics of the spirals such as length, orientation, and handedness are described. Most spirals are dorsal and comprise only a few loops. Helicomery is formed by a shift of cells during development or in adults caused by changes in cell adhesion or mechanical impacts such as lesions. A model for the formation of helicomery is proposed, which is based on medieval church labyrinths. These complex spiral structures are derived from concentric lines by the shift of relatively few tiles. This principle of "small causes, great effect" also applies to "spiral segments," because helicomery dissolves segmental patterns and questions the concept of segments as distinct structures. The widespread occurrence of helicomery in nonhomologous serial structures might indirectly indicate an underlying principle of seriality among Bilateria.

Phylogenomic Resolution of Sea Spider Diversification through Integration of Multiple Data Classes.

Despite significant advances in invertebrate phylogenomics over the past decade, the higher-level phylogeny of Pycnogonida (sea spiders) remains elusive. Due to the inaccessibility of some small-bodied lineages, few phylogenetic studies have sampled all sea spider families. Previous efforts based on a handful of genes have yielded unstable tree topologies. Here, we inferred the relationships of 89 sea spider species using targeted capture of the mitochondrial genome, 56 conserved exons, 101 ultraconserved elements, and 3 nuclear ribosomal genes. We inferred molecular divergence times by integrating morphological data for fossil species to calibrate 15 nodes in the arthropod tree of life. This integration of data classes resolved the basal topology of sea spiders with high support. The enigmatic family Austrodecidae was resolved as the sister group to the remaining Pycnogonida and the small-bodied family Rhynchothoracidae as the sister group of the robust-bodied family Pycnogonidae. Molecular divergence time estimation recovered a basal divergence of crown group sea spiders in the Ordovician. Comparison of diversification dynamics with other marine invertebrate taxa that originated in the Paleozoic suggests that sea spiders and some crustacean groups exhibit resilience to mass extinction episodes, relative to mollusk and echinoderm lineages.

A postlarval instar of Phoxichilidium femoratum (Pycnogonida, Phoxichilidiidae) with an exceptional malformation.

Individuals of the marine chelicerate lineage Pycnogonida (sea spiders) show considerable regenerative capabilities after appendage injury or loss. In their natural habitats, especially the long legs of sea spiders are commonly lost and regenerated, as is evidenced by the frequent encounter of specimens with missing or miniature legs. In contrast to this, the collection of individuals with abnormally developed appendages or trunk regions is comparably rare. Here, we studied a remarkable malformation in a postlarval instar of the species Phoxichilidium femoratum (Rathke, 1799) and describe the external morphology and internal organization of the specimen using a combination of fluorescent histochemistry and scanning electron microscopy. The individual completely lacks the last trunk segment with leg pair 4 and the normally penultimate trunk segment bears only a single aberrant appendage resembling an extension of the anteroposterior body axis. Externally, the proximal units of the articulated appendage are unpaired, but further distally a bifurcation into two equally developed leg-like branches is found. Three-dimensional reconstruction of the musculature reveals components of two regular leg muscle sets in several of the proximal articles. This confirms interpretation of the entire appendage as a malformed leg and reveals an externally hidden paired organization along its entire proximodistal axis. To explain the origin of this unique malformation, early pioneering studies on the regenerative potential of pycnogonids are evaluated and (a) an injury-induced partial fusion of the developing limb buds of leg pair 3, as well as (b) irregular leg regeneration following near complete loss of trunk segments 3 and 4 are discussed. Which of the two hypotheses is more realistic remains to be tested by dedicated experimental approaches. These will have to rely on pycnogonid species with established laboratory husbandry in order to overcome the limitations of the few short-term regeneration studies performed to date.

Aristotle's lobster: the image in the text.

The Anatomai, a lost work written by Aristotle, must have contained a collection of various drawings and figures of species as well as their organs. In his texts (mainly the Historia animalium), Aristotle is often referring to the drawings after the description of species. Our study applies the method of the comparative view ('Vergleichendes Sehen') to provide an access to and reconstruction of Aristotle's lost illustrations based on his textual descriptions. As an example, we chose the treatment of the European lobster (Homarus gammarus L., 1758) in the Aristotelian corpus as a case study. First, we analyse the etymology of the Greek term astakós referring to the lobster and provide an overview on the putative synonyms. Second, we confront the textual basis of the description with several questions concerning the degree of abstraction, the relation between text and image, and the spatial orientation of the image. Finally, we present a step-by-step reconstruction of Aristotle's illustrations of the lobster based on the various passages dealing with its anatomy in the text of the Historia animalium. The problems which arise by a confrontation of the textual basis with hypothetical images are discussed at a more general level. We conclude that this kind of a text-based image reconstruction is only possible if the object described by Aristotle is unambiguously identifiable and still visually accessible.

Eocarcinus praecursor Withers, 1932 (Malacostraca, Decapoda, Meiura) is a stem group brachyuran.

Beginning with the description by Withers in 1932, Eocarcinus praecursor from the Jurassic has long been considered the oldest representative of the Brachyura. In 2010 Feldmann and Schweitzer re-investigated the specimens of E. praecursor and expressed doubts about the brachyuran nature of this species. Among other characters, the suspected existence of small chelae in the 2nd or 3rd pereopods led them to the conclusion that E. praecursor must be removed from the Brachyura and rather be seen as a representative of the Anomala. However, Anomala also do not possess chelae on the 2nd and 3rd pereopods. This contradiction and other aspects initiated a new investigation of E. praecursor. It can be shown that neither the 2nd nor the 3rd pereopods of E. praecursor are chelate. Furthermore, there are no other derived characters shared with anomalans. By contrast, there are a number of apomorphies shared with Brachyura such as the shape and articulation of the large chelae and the attachment points of the last two pereopods. However, not all apomorphies of the crown group are present yet. Thus, E. praecursor is a stem group representative, which allows statements about individual steps in the evolution of the set of characters of the crown group Brachyura.

Comparative analysis of the antennae of three amphipod species with different lifestyles.

Crustaceans detect chemical stimuli in the environment with aesthetasc sensilla, which are located on their 1st antennae. With the transition to other environments, chemoreception faces physical challenges. To provide a deeper understanding of the relation between the morphology of olfactory organs and different lifestyles, we studied the peripheral olfactory system of three amphipod species, the marine Gammarus salinus, the blind subterranean freshwater species Niphargus puteanus, and the terrestrial Cryptorchestia garbinii. We compared the 1st and 2nd antennae of these species with respect to length and presence of aesthetascs and other sensilla. The females of N. puteanus reveal the longest 1st antennae in relation to body size. G. salinus shows the largest aesthetascs and the same relative length of the 1st antennae as male N. puteanus. C. garbinii has very short 1st antennae and reduced (putative) aesthetascs. Our findings show that the compensation of vision loss by olfaction cannot be generally assumed in animals from dark environments. Furthermore, the behaviour of C. garbinii indicates a chemosensory ability, despite the reduction of the 1st antennae. A comparison with other terrestrial crustaceans suggests that the loss of the olfactory sense on the 1st antennae in C. garbinii might be compensated with chemoreception by the 2nd antennae.

The cleavage pattern of calanoid copepods-a case study.

Many crustacean groups show stereotyped cleavage patterns during early ontogeny. However, these patterns differ between the various major crustacean taxa, and a general mode is difficult to extract. Previous studies suggested that also copepods undergo an early cleavage with a more or less stereotyped pattern of blastomere divisions and fates. Yet, copepod embryology has been largely neglected. The last investigation of this kind dates back more than a century and the results are somewhat contradictory when compared with those of other researchers. To overcome these problems, we studied the early development of a so far undescribed calanoid copepod species, Skistodiaptomus sp., applying histochemical staining, confocal laser scanning microscopy, and bifocal 4D microscopy. The blastomere arrangement of the four-cell stage of this species varies to a large degree. It can either form a typical radial pattern with the four blastomeres lying in one plane or a tilted orientation of the axes connecting the sister cells of the previous division. In both cases, a stereotyped division pattern is maintained inside each quadrant during subsequent cleavages. In addition, we found two types of blastomere arrangements with a mirror symmetry. Most divisions within the quadrants follow the perpendicularity rule until the eighth cleavage. Deviations from this rule occur only in the narrow regions where the different quadrants touch and near the site of gastrulation. Gastrulation is initiated around the descendants of one individually identifiable blastomere of the 16-cell stage. This cell divides in a specific manner forming a characteristic cell arrangement, the gastrulation triangle. This gastrulation triangle initiates the internalization process of the gastrulation and it is encircled by another characteristic cell type, the crown cells. Our observations reveal several similarities to the early development of Calanus finmarchicus, another calanoid species. These relate to blastomere arrangements and divisions and the pattern of gastrulation. As Calanoida represent a basal or near basal branch of the copepod tree, this description will provide the ground for reconstruction of the cleavage pattern of the last common ancestor of Copepoda.

Trilobite compound eyes with crystalline cones and rhabdoms show mandibulate affinities.

Most knowledge about the structure, function, and evolution of early compound eyes is based on investigations in trilobites. However, these studies dealt mainly with the cuticular lenses and little was known about internal anatomy. Only recently some data on crystalline cones and retinula cells were reported for a Cambrian trilobite species. Here, we describe internal eye structures of two other trilobite genera. The Ordovician Asaphus sp. reveals preserved crystalline cones situated underneath the cuticular lenses. The same is true for the Devonian species Archegonus (Waribole) warsteinensis, which in addition shows the fine structure of the rhabdom in the retinula cells. These results suggest that an apposition eye with a crystalline cone is ancestral for Trilobita. The overall similarity of trilobite eyes to those of myriapods, crustaceans, and hexapods corroborates views of a phylogenetic position of trilobites in the stem lineage of Mandibulata.

Segmentation and limb formation during naupliar development of Tigriopus californicus (Copepoda, Harpacticoida).

Naupliar development in copepods includes the generation of usually five pairs of post-mandibular segments. Since copepod nauplii show no outer body articulation, the only indication of larval segmentation is the expression of limb buds. Yet, in copepods the timing and sequence of limb bud expression in larval development varies to a large degree. In harpacticoid nauplii for instance, the 1st maxillae are formed at an early naupliar stage. By contrast, the four remaining pairs of limb buds frequently appear simultaneously with the last naupliar stage. The complete process of larval segment formation takes place under the body surface and has never been described in detail. To broaden our knowledge of early segmentation in copepods, we here describe the segmentation of the harpacticoid nauplius Tigriopus californicus by analysing the expression of the segment marker Engrailed and uncover the sequential addition of seven post-mandibular segments. The stripe formation and arrangement of labelled cells corresponds largely to those of other crustaceans studied in this respect. Together with a morphological approach using histology, SEM, and 3D-reconstructions based on CLSM we solve the so far controversial identity of the external limb buds in the final naupliar stage. In contrast to previous studies, we can show that all limb pairs from the 1st maxillae to the 3rd thoracopods are formed. Yet, the anlage of the maxilliped (1st thoracopod) remains hidden underneath the cuticle being never externally expressed in the nauplius.

Adaptation Aftereffects in the Perception of Crabs and Lobsters as Examples of Complex Natural Objects.

To recognize a familiar object, incoming perceptual information is matched against object representations in memory. Mounting evidence suggests that these representations are not stable, but adapt flexibly to recently encountered perceptual information. This is evident in the form of aftereffects, where prolonged exposure to one object (adaptor) influences perception of the next (test stimulus). So far, adaptation aftereffects have been mainly shown for human faces and simple geometric shapes, and it has been concluded that face aftereffects partially derive from shape adaptation. However, it is largely unknown whether adaptation aftereffects generalize to other categories of complex, naturalistic biological objects, and if so, whether these effects can be explained by shape adaptation. To answer these questions, we conducted three experiments in which images of crabs and lobsters were presented in two versions: as complex, naturalistic images, or reduced to their simplified geometric shapes. In Experiment 1, we found robust adaptation aftereffects for the complex versions of the images, indicating that adaptation aftereffects generalize to animate objects other than faces. Experiment 2 showed adaptation aftereffects for the simplified stimuli, replicating previous findings on geometric shapes. Experiment 3 demonstrated that adaptation to the simplified animal shapes results in aftereffects on the complex naturalistic stimuli. Comparisons between experiments revealed that aftereffects were largest in the first experiment, in which complex stimuli served as adaptor and test stimuli. Together, these experiments show that the magnitude of adaptation aftereffects depends on the complexity of the adaptor, but not on that of the test stimuli, and that shape adaptation plays a role in - but cannot entirely account for - the object aftereffects.

The reproductive system of Limnopilos naiyanetri indicates a thoracotreme affiliation of Hymenosomatidae (Decapoda, Eubrachyura).

The eubrachyuran Hymenosomatoidea is widely distributed in tropical and subtropical regions ranging from marine to freshwater habitats. Even though the biology of this taxon has been studied to some extent, its phylogenetic relationships are not resolved. Based on different morphological characters, some authors suggested a close affinity of hymenosomatid crabs to heterotremes. However, many of these characters are ambiguous, and the few molecular studies did not provide convincing solutions either. To address this issue, we studied the reproductive system of the hymenosomatid freshwater species Limnopilos naiyanetri Chuang and Ng, 1991 using histology and scanning electron microscopy. The females show the characteristic organization of the paired eubrachyuran reproductive system. Additionally, a bursa (an accessory sperm storing cuticle cavity) is present. The male copulatory system is characterized by paired long first and short second gonopods, and a pair of sternal gonopores equipped with a penis. Both, the female and male reproductive organs reveal a number of similarities to thoracotreme crabs. The seminal receptacle is lined by a very thin cuticle and by a mono-layered glandular epithelium. The male gonopods and the sternal genital opening also resemble the thoracotreme condition. Thus, our results indicate that Hymenosomatidae are most likely part of the Thoracotremata.

Comparison of ventral organ development across Pycnogonida (Arthropoda, Chelicerata) provides evidence for a plesiomorphic mode of late neurogenesis in sea spiders and myriapods.

BACKGROUND: Comparative studies of neuroanatomy and neurodevelopment provide valuable information for phylogenetic inference. Beyond that, they reveal transformations of neuroanatomical structures during animal evolution and modifications in the developmental processes that have shaped these structures. In the extremely diverse Arthropoda, such comparative studies contribute with ever-increasing structural resolution and taxon coverage to our understanding of nervous system evolution. However, at the neurodevelopmental level, in-depth data remain still largely confined to comparably few laboratory model organisms. Therefore, we studied postembryonic neurogenesis in six species of the bizarre Pycnogonida (sea spiders), which - as the likely sister group of all remaining chelicerates - promise to illuminate neurodevelopmental changes in the chelicerate lineage. RESULTS: We performed in vivo cell proliferation experiments with the thymidine analogs 5-bromo-2'-deoxyuridine and 5-ethynl-2'-deoxyuridine coupled to fluorescent histochemical staining and immunolabeling, in order to compare ventral nerve cord anatomy and to localize and characterize centers of postembryonic neurogenesis. We report interspecific differences in the architecture of the subesophageal ganglion (SEG) and show the presence of segmental "ventral organs" (VOs) that act as centers of neural cell production during gangliogenesis. These VOs are either incorporated into the ganglionic soma cortex or found on the external ganglion surface. Despite this difference, several shared features support homology of the two VO types, including (1) a specific arrangement of the cells around a small central cavity, (2) the presence of asymmetrically dividing neural stem cell-like precursors, (3) the migration of newborn cells along corresponding pathways into the cortex, and (4) the same VO origin and formation earlier in development. CONCLUSIONS: Evaluation of our findings relative to current hypotheses on pycnogonid phylogeny resolves a bipartite SEG and internal VOs as plesiomorphic conditions in pycnogonids. Although chelicerate taxa other than Pycnogonida lack comparable VOs, they are a characteristic feature of myriapod gangliogenesis. Accordingly, we propose internal VOs with neurogenic function to be part of the ground pattern of Arthropoda. Further, our findings illustrate the importance of dense sampling in old arthropod lineages - even if as gross-anatomically uniform as Pycnogonida - in order to reliably differentiate plesiomorphic from apomorphic neurodevelopmental characteristics prior to outgroup comparison.

Larval neurogenesis in the copepod Tigriopus californicus (Tetraconata, Multicrustacea).

Arthropod early neurogenesis shows distinct patterns that have been interpreted in an evolutionary framework. For instance, crustaceans and Hexapoda form the taxon Tetraconata and share the differentiation of specific neural precursors, the neuroblasts, a character which sets them apart from Chelicerata and Myriapoda. Neuroblasts are relatively large stem cells that generate ganglion mother cells by asymmetric divisions. Ganglion mother cells typically divide once to give rise to neurons and glia cells. In hexapods, neuroblasts segregate from the neuroectoderm before they begin their characteristic proliferative activity. In the crustaceans studied so far, neuroblasts remain in the neuroectoderm. Yet, detailed studies on early neurogenesis of crustaceans at the cellular level are largely restricted to some malacostracan and branchiopod species. Crustaceans are very diverse and likely paraphyletic with respect to hexapods. Hence, knowledge about neural differentiation in other crustacean taxa might contribute to the understanding of evolution of neurogenesis in Tetraconata. Here, we describe the early neurogenesis during naupliar development of the copepod Tigriopus californicus. We show that neuroblasts are present that generate ganglion mother cells, which in turn divide to give rise to neurons of the ventral nerve cord. These two neural precursor cell types and their specific arrangement correspond to what has been found in other crustaceans. One obvious difference concerns the relative size of the neuroblasts, which are not much larger than their progeny. Our results complement the picture of neural differentiation in crustaceans and suggest that superficially located neuroblasts are likely the ancestral condition in Tetraconata.