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Abstract Background Two types of excretory systems, protonephridia and metanephridial systems are common among bilaterians. The homology of protonephridia of lophotrochozoan taxa has been widely accepted. In contrast, the homology of metanephridial systems – including coelomic cavities as functional units – among taxa as well as the homology between the two excretory systems is a matter of ongoing discussion. This particularly concerns the molluscan kidneys, which are mostly regarded as being derived convergently to the metanephridia of e.g. annelids because of different ontogenetic origin. A reinvestigation of nephrogenesis in polyplacophorans, which carry many primitive traits within molluscs, could shed light on these questions. Results The metanephridial system of Lepidochitona corrugata develops rapidly in the early juvenile phase. It is formed from a coelomic anlage that soon achieves endothelial organization. The pericardium and heart are formed from the central portion of the anlage. The nephridial components are formed by outgrowth from lateral differentiations of the anlage. Simultaneously with formation of the heart, podocytes appear in the atrial wall of the pericardium. In addition, renopericardial ducts, kidneys and efferent nephroducts, all showing downstream ciliation towards the internal lumen, become differentiated (specimen length: 0.62 mm). Further development consists of elongation of the kidney and reinforcement of filtration and reabsorptive structures. Conclusions During development and in fully formed condition the metanephridial system of Lepidochitona corrugata shares many detailed traits (cellular and overall organization) with the protonephridia of the same species. Accordingly, we suggest a serial homology of various cell types and between the two excretory systems and the organs as a whole. The formation of the metanephridial system varies significantly within Mollusca, thus the mode of formation cannot be used as a homology criterion. Because of similarities in overall organization, we conclude that the molluscan metanephridial system is homologous with that of the annelids not only at the cellular but also at the organ level.

In this study, we apply a two-step (untreated and soft tissue stained) diffusible iodine-based contrast-enhanced micro-computed tomography array to a wet-collection Lantern Shark specimen of Etmopterus lucifer. The focus of our scanning approach is the head anatomy. The unstained CT data allow the imaging of mineralized (skeletal) tissue, while results for soft tissue were achieved after staining for 120 h in a 1% ethanolic iodine solution. Three-dimensional visualization after the segmentation of hard as well as soft tissue reveals new details of tissue organization and allows us to draw conclusions on the significance of organs in their function. Outstanding are the ampullae of Lorenzini for electroreception, which appear as the dominant sense along with the olfactory system. Corresponding brain areas of these sensory organs are significantly enlarged as well and likely reflect adaptations to the lantern sharks' deep-sea habitat. While electroreception supports the capture of living prey, the enlarged olfactory system can guide the scavenging of these opportunistic feeders. Compared to other approaches based on the manual dissection of similar species, CT scanning is superior in some but not all aspects. For example, fenestrae of the cranial nerves within the chondrocranium cannot be identified reflecting the limitations of the method, however, CT scanning is less invasive, and the staining is mostly reversible and can be rinsed out., (© 2023 The Authors. Journal of Anatomy published by John Wiley & Sons Ltd on behalf of Anatomical Society.)

Our understanding of metabolic interactions between small symbiotic animals and bacteria or parasitic eukaryotes that reside within their bodies is extremely limited. This gap in knowledge originates from a methodological challenge, namely to connect histological changes in host tissues induced by beneficial and parasitic (micro)organisms to the underlying metabolites. We addressed this challenge and developed chemo-histo-tomography (CHEMHIST), a culture-independent approach to connect anatomic structure and metabolic function in millimeter-sized symbiotic animals. CHEMHIST combines chemical imaging of metabolites based on mass spectrometry imaging (MSI) and microanatomy-based micro-computed X-ray tomography (micro-CT) on the same animal. Both high-resolution MSI and micro-CT allowed us to correlate the distribution of metabolites to the same animal's three-dimensional (3D) histology down to submicrometer resolutions. Our protocol is compatible with tissue-specific DNA sequencing and fluorescence in situ hybridization for the taxonomic identification and localization of the associated micro(organisms). Building CHEMHIST upon in situ imaging, we sampled an earthworm from its natural habitat and created an interactive 3D model of its physical and chemical interactions with bacteria and parasitic nematodes in its tissues. Combining MSI and micro-CT, we present a methodological groundwork for connecting metabolic and anatomic phenotypes of small symbiotic animals that often represent keystone species for ecosystem functioning., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Primitive cnidarians are crucial for elucidating the early evolution of metazoan body plans and life histories in the late Neoproterozoic and Palaeozoic. The highest complexity of both evolutionary aspects within cnidarians is found in extant hydrozoans. Many colonial hydrozoans coated with chitinous exoskeletons have the potential to form fossils; however, only a few fossils possibly representing hydroids have been reported, which still require scrutiny. Here, we present an exceptionally well-preserved hydroid found in the Upper Cambrian Fengshan Formation in northern China. It was originally interpreted as a problematic graptolite with an uncertain systematic position. Based on three characteristic morphological traits shared with extant hydroids (with paired hydrothecae, regular hydrocaulus internodes and special intrathecal origin pattern of hydrocladium), we propose this fossil hydroid as a new genus, Palaeodiphasia gen. nov., affiliated with the advanced monophyletic hydrozoan clade Macrocolonia typically showing loss of the medusa stage. More Macrocolonia fossils reviewed here indicate that this life strategy of medusa loss has been achieved already as early as the Middle Devonian. The early stratigraphical appearance of such advanced hydroid contrasts with previous molecular hypotheses regarding the timing of medusozoan evolution, and may be indicative for understanding the Ediacaran cnidarian radiation.

Lonchoptera lutea males produce giant spermatozoa that are more than 2000 μm long and 1.4 μm wide. Unlike the typical brachyceran spermatozoon, they have a highly asymmetrical cross-section with only a single, albeit very large, mitochondrial derivative and a pair of massive accessory bodies, one of which extends throughout the entire length of the sperm tail. The accessory bodies consist of an electron-dense matrix in which numerous peculiar electron-lucid substructures are embedded. In the mated female, the giant spermatozoa are found inside two tubular spermathecae which are also extremely long, measuring 4000 μm or more., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: The diversification process known as the Lake Tanganyika Radiation has given rise to the most speciose clade of African cichlids. Almost all cichlid species found in the lakes Tanganyika, Malawi and Victoria, comprising a total of 12-16 tribes, belong to this clade. Strikingly, all the species in the latter two lakes are members of the tribe Haplochromini, whose origin remains unclear. The 'out of Tanganyika' hypothesis argues that the Haplochromini emerged simultaneously with other cichlid tribes and lineages in Lake Tanganyika, presumably about 5-6 million years ago (MYA), and that their presence in the lakes Malawi and Victoria and elsewhere in Africa today is due to later migrations. In contrast, the 'melting pot Tanganyika hypothesis' postulates that Haplochromini emerged in Africa prior to the formation of Lake Tanganyika, and that their divergence could have begun about 17 MYA. Haplochromine fossils could potentially resolve this debate, but such fossils are extremely rare., Results: Here we present a new fossil haplochromine from the upper Miocene site Waril (9-10 million years) in Central Kenya. Comparative morphology, supported by Micro-CT imaging, reveals that it bears a unique combination of characters relating to dentition, cranial bones, caudal skeleton and meristic traits. Its most prominent feature is the presence of exclusively unicuspid teeth, with canines in the outer tooth row. †Warilochromis unicuspidatus gen. et sp. nov. shares this combination of characters solely with members of the Haplochromini and its lacrimal morphology indicates a possible relation to the riverine genus Pseudocrenilabrus. Due to its fang-like dentition and non-fusiform body, †W. unicuspidatus gen. et sp. nov. might have employed either a sit-and-pursue or sit-and-wait hunting strategy, which has not been reported for any other fossil haplochromine cichlid., Conclusions: The age of the fossil (9-10 MYA) is incompatible with the 'out of Tanganyika' hypothesis, which postulates that the divergence of the Haplochromini began only 5-6 MYA. The presence of this fossil in an upper Miocene palaeolake in the Central Kenya Rift, as well as its predatory lifestyle, indicate that Haplochromini were already an important component of freshwater drainages in East Africa at that time.

In the field of correlative microscopy, light and electron microscopy form a powerful combination for morphological analyses in zoology. Due to sample thickness limitations, these imaging techniques often require sectioning to investigate small animals and thereby suffer from various artefacts. A recently introduced nanoscopic X-ray computed tomography (NanoCT) setup has been used to image several biological objects, none that were, however, embedded into resin, which is prerequisite for a multitude of correlative applications. In this study, we assess the value of this NanoCT for correlative microscopy. For this purpose, we imaged a resin-embedded, meiofaunal sea cucumber with an approximate length of 1 mm, where microCT would yield only little information about the internal anatomy. The resulting NanoCT data exhibits isotropic 3D resolution, offers deeper insights into the 3D microstructure, and thereby allows for a complete morphological characterization. For comparative purposes, the specimen was sectioned subsequently to evaluate the NanoCT data versus serial sectioning light microscopy (ss-LM). To correct for mechanical instabilities and drift artefacts, we applied an alternative alignment procedure for CT reconstruction. We thereby achieve a level of detail on the subcellular scale comparable to ss-LM images in the sectioning plane.

Background: The astonishing variety of sounds that birds can produce has been the subject of many studies aiming to identify the underlying anatomical and physical mechanisms of sound production. An interesting feature of some bird vocalisations is the simultaneous production of two different frequencies. While most work has been focusing on songbirds, much less is known about dual-sound production in non-passerines, although their sound production organ, the syrinx, would technically allow many of them to produce "two voices". Here, we focus on the king penguin, a colonial seabird whose calls consist of two fundamental frequency bands and their respective harmonics. The calls are produced during courtship and for partner and offspring reunions and encode the birds' identity. We dissected, μCT-scanned and analysed the vocal tracts of six adult king penguins from Possession Island, Crozet Archipelago., Results: King penguins possess a bronchial type syrinx that, similarly to the songbird's tracheobronchial syrinx, has two sets of vibratory tissues, and thus two separate sound sources. Left and right medial labium differ consistently in diameter between 0.5 and 3.2%, with no laterality between left and right side. The trachea has a conical shape, increasing in diameter from caudal to cranial by 16%. About 80% of the king penguins' trachea is medially divided by a septum consisting of soft elastic tissue (septum trachealis medialis)., Conclusions: The king penguins' vocal tract appears to be mainly adapted to the life in a noisy colony of a species that relies on individual vocal recognition. The extent between the two voices encoding for individuality seems morphologically dictated by the length difference between left and right medial labium. The septum trachealis medialis might support this extent and could therefore be an important anatomical feature that aids in the individual recognition process., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s). 2020.)

The present study aims to clarify the confused taxonomy of Z.schaufussi von Frauenfeld, 1862 and Zospeumsuarezi Gittenberger, 1980. Revision of Iberian Zospeum micro snails is severely hindered by uncertainties regarding the identity of the oldest Iberian Zospeum species, Z.schaufussi von Frauenfeld, 1862. In this paper, we clarify its taxonomic status by designating a lectotype from the original syntype series and by describing its internal and external shell morphology. Using SEM-EDX, we attempt to identify the area of the type locality cave more precisely than "a cave in Spain". The shell described and illustrated by Gittenberger (1980) as Z.schaufussi appears not to be conspecific with the lectotype shell, and is considered a separate species, Z.gittenbergeri Jochum, Prieto & De Winter, sp. n. Zospeumsuarezi was described from various caves in NW Spain. Study of the type material reveals that these shells are not homogenous in shell morphology. The holotype shell of Z.suarezi is imaged here for the first time. The paratype shell, illustrated by Gittenberger (1980) from a distant, second cave, is described as Zospeumpraetermissum Jochum, Prieto & De Winter, sp. n. The shell selected here as lectotype of Z.schaufussi , was also considered a paratype of Z.suarezi by Gittenberger (1980). Since this specimen is morphologically very similar to topotypic shells of Z.suarezi , the latter species is considered a junior synonym of Z.schaufussi ( syn. n. ). The internal shell morphology of all these taxa is described and illustrated using X-ray Micro Computer Tomography (Micro-CT).

African sponges, particularly freshwater sponges, are understudied relative to demosponges in most other geographical regions. Freshwater sponges (Spongillida) likely share a common ancestor; however, their evolutionary history, particularly during their radiation into endemic and allegedly cosmopolitan groups, is unclear. Freshwater sponges of at least 58 species of 17 genera and four families are described from Central and Eastern Africa, but the diversity is underestimated due to limited distinguishable morphological features. The discovery of additional cryptic species is very likely with the use of molecular techniques such as DNA barcoding. The Royal Museum of Central Africa (MRAC, Tervuren, Belgium) hosts one of the largest collections of (Central) African freshwater sponge type material. Type specimens in theory constitute ideal targets for molecular taxonomy; however, the success is frequently hampered by DNA degradation and deamination, which are a consequence of suboptimal preservation techniques. Therefore, we genotyped African demosponge holotype material of the MRAC with specific short primers suitable for degenerated tissue and compare the results with the current, morphology-based classification. Our results demonstrate the utility of minimalistic barcodes for identification of sponges, potentially enabling efficient identification of individuals in taxonomic or metabarcoding studies, and highlight inconsistencies in the current freshwater sponge classification.

Five years ago, the Panamanian evolutionary lineage (EL) C12 was uncovered along with four other ELs in an integrative phylogenetic investigation of worldwide Carychiidae. Since EL C12 lacked shell material post-molecular analysis to serve as a museum voucher, it remained undescribed. Now, after recent collection efforts of C12 and the congener, Carychiumzarzaae Jochum & Weigand, 2017 at their original Panamanian sites, C12 is morphologically described and formally assigned the name, Carychiumpanamaense Jochum, sp. n. In sync with recent taxonomic treatment of the genus, computed tomography (CT) is used in this work to differentiate shells of C.panamaense sp. n. from geographically-proximal, Caribbean, North and Central American congeners. Recent material of topotypic Carychiumjardineanum (Chitty, 1853) and undamaged C.zarzaae were additionally CT-scanned and assessed in the comparative analyses.

Cryptic species have been detected in many groups of organisms and must be assumed to make up a significant portion of global biodiversity. We study geckos of the Ebenavia inunguis complex from Madagascar and surrounding islands and use species delimitation algorithms (GMYC, BOLD, BPP), COI barcode divergence, diagnostic codon indels in the nuclear marker PRLR, diagnostic categorical morphological characters, and significant differences in continuous morphological characters for its taxonomic revision. BPP yielded ≥ 10 operational taxonomic units, whereas GMYC (≥ 27) and BOLD (26) suggested substantial oversplitting. In consequnce, we resurrect Ebenavia boettgeri Boulenger 1885 and describe Ebenavia tuelinae sp. nov., Ebenavia safari sp. nov., and Ebenavia robusta sp. nov., increasing the number of recognised species in Ebenavia from two to six. Further lineages of Ebenavia retrieved by BPP may warrant species or subspecies status, but further taxonomic conclusions are postponed until more data become available. Finally, we present an identification key to the genus Ebenavia, provide an updated distribution map, and discuss the diagnostic values of computational species delimitation as well as morphological and molecular diagnostic characters.

Metastatic seeding is driven by cell-intrinsic and environmental cues, yet the contribution of biomechanics is poorly known. We aim to elucidate the impact of blood flow on the arrest and the extravasation of circulating tumor cells (CTCs) in vivo. Using the zebrafish embryo, we show that arrest of CTCs occurs in vessels with favorable flow profiles where flow forces control the adhesion efficacy of CTCs to the endothelium. We biophysically identified the threshold values of flow and adhesion forces allowing successful arrest of CTCs. In addition, flow forces fine-tune tumor cell extravasation by impairing the remodeling properties of the endothelium. Importantly, we also observe endothelial remodeling at arrest sites of CTCs in mouse brain capillaries. Finally, we observed that human supratentorial brain metastases preferably develop in areas with low perfusion. These results demonstrate that hemodynamic profiles at metastatic sites regulate key steps of extravasation preceding metastatic outgrowth., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The diversity and phylogenetic relationships of calcareous sponges are still not completely understood. Recent integrative approaches combined analyses of DNA and morphological observations. Such studies resulted in severe taxonomic revisions within the subclass Calcinea and provided the foundation for a phylogenetically meaningful classification. However, several genera are missing from DNA phylogenies and their relationship to other Calcinea remain uncertain. One of these genera is Leuclathrina (family Leucaltidae). We here describe a new species from the Maldives, Leuclathrina translucida sp. nov., which is only the second species of the genus. Like the type species Leuclathrina asconoides, the new species has a leuconoid aquiferous system and lacks a specialized choanoskeleton. Phylogenetic analyses of the partial 28S ribosomal RNA gene revealed that L. translucida sp. nov. is most closely related to a clade containing the exclusively asconoid genera Ascandra, Levinella and Soleneiscus, and to a clade of the likewise asconoid genus Ernstia. No close relationship exists to other members of the polyphyletic family Leucaltidae, or to any other leuconoid Calcinea. Our results suggest that the leuconoid aquiferous system of Leuclathrina evolved independently from that of other calcineans and that the family assignment of the genus has to be reconsidered. Because the latter requires a more comprehensive family level revision among many genera of Calcinea, we propose to formally retain the genus in Leucaltidae for the time being.

The rapid and synchronous divisions of large and transparent oocytes, eggs, and embryos of marine species are exceptionally well suited for microscopic observation. Consequently, these cells have been models for cell division research since its beginnings and contributed some of its first and most fundamental discoveries. While large size and rapid transitions render these cells ideal specimens for light microscopy, the same features constitute a challenge for electron microscopy. Here, we describe example protocols from our work on starfish oocyte meiosis, where we overcome these challenges by using live imaging of fluorescently labeled structures in combination with correlated electron microscopy. In this work, we demonstrate how: (i) to capture a rapid, transient event in time and (ii) to localize a small structure within the large oocyte. These techniques are applicable with minor modifications to oocytes and embryos of other species and, possibly, to other cell types., (© 2018 Elsevier Inc. All rights reserved.)

Aggregation is a common process occurring in many diverse particulate gas mixtures (e.g. those derived from explosive volcanic eruptions, meteorite impact events, and fluid bed processing). It results from the collision and sticking of particles suspended in turbulent gas/air. To date, there is no generalized model of the underlying physical processes. Here, we investigate aggregates from 18 natural deposits (16 volcanic deposits and two meteorite impact deposits) as well as aggregates produced experimentally via fluidized bed techniques. All aggregates were analyzed for their size, internal structuring, and constituent particle size distribution. Commonalities and differences between the aggregate types are then used to infer salient features of the aggregation process. Average core to rim ratios of internally structured aggregates (accretionary lapilli) is found to be similar for artificial and volcanic aggregates but up to an order of magnitude different than impact-related aggregates. Rim structures of artificial and volcanic aggregates appear to be physically similar (single, sub-spherical, regularly-shaped rims) whereas impact-related aggregates more often show multiple or irregularly shaped rims. The particle size distributions (PSDs) of all three aggregate types are similar (< 200 μm). This proves that in all three environments, aggregation occurs under broadly similar conditions despite the significant differences in source conditions (particle volume fraction, particle size distribution, particle composition, temperature), residence times, plume conditions (e.g., humidity and temperature), and dynamics of fallout and deposition. Impact-generated and volcanic aggregates share many similarities, and in some cases may be indistinguishable without their stratigraphic context.

Amblyomma birmitum sp. nov. is formally described as a new record from 99 Ma old Burmese amber from Myanmar. This confirms the presence of the extant hard tick genus Amblyomma C.L. Koch, 1844 (Ixodida: Ixodidae) in the Late Cretaceous. This discovery is placed in its wider context and some reports of fossil hard ticks, such as a Hyalomma C.L. Koch, 1844 in Eocene Baltic amber, are misidentifications. The genus Amblyomma belongs to the clade Metastriata, a group which probably also accommodates two extinct genera, Cornupalpatum Poinar and Brown, 2003 and Compluriscutata Poinar and Buckley, 2008, also found in Burmese amber. All three fossils are thus only a little younger than published molecular divergence time estimates (ca. 124 ± 17 Ma) for the Metastriata lineage. Amblyomma has a largely Gondwanan distribution today. However, in some biogeographical scenarios, e.g. the Samafrica model, its predicted radiation time postdates the dissolution of the original Gondwana supercontinent raising questions about how its current distribution pattern was achieved.

Three new species of the genus Carychium O.F. Müller, 1773, Carychium hardiei Jochum & Weigand, sp. n. , Carychium belizeense Jochum & Weigand, sp. n. and Carychium zarzaae Jochum & Weigand, sp. n. are described from the Southeastern United States, Belize and Panama, respectively. In two consecutive molecular phylogenetic studies of worldwide members of Carychiidae, the North and Central American morphospecies Carychium mexicanum Pilsbry, 1891 and Carychium costaricanum E. von Martens, 1898 were found to consist of several evolutionary lineages. Although the related lineages were found to be molecularly distinct from the two nominal species, the consequential morphological and taxonomic assessment of these lineages is still lacking. In the present paper, the shells of these uncovered Carychium lineages are assessed by comparing them with those of related species, using computer tomography for the first time for this genus. The interior diagnostic characters are emphasized, such as columellar configuration in conjunction with the columellar lamella and their relationship in context of the entire shell. These taxa are morphologically described and formally assigned their own names.

Combining in vivo imaging with electron microscopy (EM) uniquely allows monitoring rare and critical events in living tissue, followed by their high-resolution visualization in their native context. A major hurdle, however, is to keep track of the region of interest (ROI) when moving from intravital microscopy (IVM) to EM. Here, we present a workflow that relies on correlating IVM and microscopic X-ray computed tomography to predict the position of the ROI inside the EM-processed sample. The ROI can then be accurately and quickly targeted using ultramicrotomy and imaged using EM. We outline how this procedure is used to retrieve and image tumor cells arrested in the vasculature of the mouse brain., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The morphological character of the hydrocladium and gonotheca origin from within the hydrothecal cavity has rarely been applied for generic diagnoses in hydrozoans. Its taxonomic value has been controversial for more than a century. The genus Fraseroscyphus Boero and Bouillon, 1993 (Hydrozoa: Sertulariidae) is a relatively recently debated case and it has been distinguished from Symplectoscyphus Marktanner-Turneretscher, 1890 based on this character. A review of this character in all published nominal species of the family Sertulariidae reveals that its occurrence is inconsistent at the genus level. However, phylogenetic analyses based on mitochondrial (16S) and nuclear (18S, 28S) genes support the position of Fraseroscyphus as a genus within the family Symplectoscyphidae Maronna et al., 2016. Comparisons of 16 morphological characters of 10 related species support the distinction of Fraseroscyphus from Antarctoscyphus and Symplectoscyphus by other characters in addition to the hydrocladial and gonothecal origin character. These new characters include the rarely-branched hydrocaulus, the absence of an apophysis, and the absence of an axillary hydrotheca. Furthermore, a revision based on the morphological character complex mentioned above using type and topotypic material, demonstrated that Sertularella sinuosa Fraser, 1948 (type species of Fraseroscyphus) and Symplectoscyphus huanghaiensis Tang & Huang, 1986 are junior synonyms of F. hozawai (Stechow, 1931) comb. nov. The assignment of Sertularella irregularis Trebilcock, 1928 and Sertularella macrogona Trebilcock, 1928 to Fraseroscyphus is also supported. In addition, sequence polymorphism of mitochondrial genes even within a single hydroid fragment was detected by the molecular cloning method, and is probably in part attributable to errors introduced by PCR, mitochondrial heteroplasmy and/or nuclear mitochondrial DNA (NUMTs). The adoption of the cloning method may be crucial to improve the sequence accuracy for some colonial hydrozoans.

Intravital microscopy provides dynamic understanding of multiple cell biological processes, but its limited resolution has so far precluded structural analysis. Because it is difficult to capture rare and transient events, only a few attempts have been made to observe specific developmental and pathological processes in animal models using electron microscopy. The multimodal correlative approach that we propose here combines intravital microscopy, microscopic X-ray computed tomography and three-dimensional electron microscopy. It enables a rapid (c.a. 2 weeks) and accurate (<5 µm) correlation of functional imaging to ultrastructural analysis of single cells in a relevant context. We demonstrate the power of our approach by capturing single tumor cells in the vasculature of the cerebral cortex and in subcutaneous tumors, providing unique insights into metastatic events. Providing a significantly improved throughput, our workflow enables multiple sampling, a prerequisite for making correlative imaging a relevant tool to study cell biology in vivo. Owing to the versatility of this workflow, we envision broad applications in various fields of biological research, such as cancer or developmental biology., (© 2016. Published by The Company of Biologists Ltd.)

Background: To date only one species of Limbodessus diving beetles has been reported from the Island of New Guinea, L. compactus (Clark, 1862), which is widerspread in the Australian region., New Information: We describe two new species of microendemic New Guinea Limbodessus and use a compact descriptive format flanked by enriched online content in wiki powered species pages. Limbodessus baliem sp.n. is described from ca. 1,600 m altitude in the Baliem Valley of Papua and Limbodessus alexanderi sp.n. from >3,000 m altitude north of Sugapa, Papua. Based on our analysis, we also transfer three species from other genera to Limbodessus Guignot, 1939, with the following changes: Limbodessus deflectus (Ordish, 1966), new combination; Limbodessus leveri (J. Balfour-Browne, 1944), new combination; and Limbodessus plicatus (Sharp, 1882), new combination.

We revise the taxonomic status of two species of Madagascan chameleons in light of a recent molecular phylogenetic study on the Calumma nasutum group. The investigation of morphological and osteological characters led to a clear delineation between two species within the C. boettgeri complex, C. boettgeri and C. linotum. Calumma linotum has been considered either a synonym of C. boettgeri or a dubious, poorly defined taxon. So far it has only been known from the male holotype with the imprecise locality 'Madagascar'. Based on pholidosis, morphological measurements and characters of the skull that were analyzed using micro-X-ray computed tomography (micro-CT) scans, we ascribe the population of chameleons from Montagne d'Ambre, formerly assigned to C. boettgeri, to C. linotum. Calumma linotum differs from C. boettgeri in the larger size of tubercle scales on the extremities and rostral appendage, the larger diameter of the extremities relative to the body size, the presence of a parietal crest as well as the form of the nasal bones and the anterior tip of the frontal. The life colouration of the males is also characteristic, with a blue rostral appendage and greenish turquoise extremities. The body and rostral appendage of C. boettgeri in contrast are inconspicuously yellowish brown coloured. All confirmed distribution records of C. boettgeri are confined to the biogeographic Sambirano region whereas C. linotum is only known from Montagne d'Ambre and a locality at the base of the Tsaratanana massif. Additional literature records of C. boettgeri and C. linotum from northeastern Madagascar are in need of confirmation. We also confirm the synonymy of Chamaeleo macrorhinus (described from a female holotype with an unknown locality) with Calumma boettgeri. The use of micro-CT exposed further characteristics for species delimitation in an integrative taxonomic approach. In addition to the skull, we also micro-CT scanned the hemipenes of C. boettgeri and C. linotum, using an iodine-based tissue stain, and provide 3D PDF models of these organs. This method enables detailed illustration and the detection of variation in particular characters, and might be an important tool in further taxonomic studies on the C. nasutum group and other squamate reptiles.

The rainforests of the Marojejy massif in northern Madagascar are a well-known hotspot of amphibian species diversity and endemism. In the present paper, we re-describe Rhombophryne serratopalpebrosa (Guibé 1975), a cophyline microhylid frog from high altitude on this massif, based on a re-examination of its holotype, and describe Rhombophryne vaventy sp. nov. using characters of external morphology and osteology, illustrated by pdf-embedded comparative 3D models of their skeletons. Rhombophryne serratopalpebrosa differs from R. vaventy sp. nov. by smaller size (28 mm snout-vent length vs. 52.9 mm), skin texture (granular vs. rough and tubercular skin respectively), supratympanic fold shape (strong, long and straight reaching the eye vs. curved and not extending anteriorly beyond the tympanum), relative tympanum diameter (78% vs. 41% of eye diameter), shape of the postchoanal prevomerine palate, shape of the footplate of the columella, length of prepollex, and by other subtle osteological features. Morphological comparisons suggest that a specimen from Ambolokopatrika assigned to R. serratopalpebrosa in previous genetic studies might belong to yet another undescribed species, closely related to R. vaventy sp. nov., whereas DNA sequences of the topotypic R. serratopalpebrosa remain unknown. We therefore emphasise the need for collecting additional material from high altitudes of the Marojejy massif to understand the systematics, as well as the natural history, of this poorly known species. For the new species described herein, we propose a Red List threat status of Vulnerable, in line with other Marojejy endemics from a similar altitude.

With the latest release of the S2PLOT graphics library, embedding interactive, 3-dimensional (3-d) scientific figures in Adobe Portable Document Format (PDF) files is simple, and can be accomplished without commercial software. In this paper, we motivate the need for embedding 3-d figures in scholarly articles. We explain how 3-d figures can be created using the S2PLOT graphics library, exported to Product Representation Compact (PRC) format, and included as fully interactive, 3-d figures in PDF files using the movie15 LaTeX package. We present new examples of 3-d PDF figures, explain how they have been made, validate them, and comment on their advantages over traditional, static 2-dimensional (2-d) figures. With the judicious use of 3-d rather than 2-d figures, scientists can now publish, share and archive more useful, flexible and faithful representations of their study outcomes. The article you are reading does not have embedded 3-d figures. The full paper, with embedded 3-d figures, is recommended and is available as a supplementary download from PLoS ONE (File S2).

Background: In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information. Here we demonstrate the advantages of the correlative use of microCT, light microscopy, and transmission electron microscopy for the analysis of small biological samples., Results: We used a small juvenile bivalve mollusc (Mytilus galloprovincialis, approximately 0.8 mm length) to demonstrate the workflow of a correlative examination by microCT, LM serial section analysis, and TEM-re-sectioning. Initially these three datasets were analyzed separately, and subsequently they were fused in one 3D scene. This workflow is very straightforward. The specimen was processed as usual for transmission electron microscopy including post-fixation in osmium tetroxide and embedding in epoxy resin. Subsequently it was imaged with microCT. Post-fixation in osmium tetroxide yielded sufficient X-ray contrast for microCT imaging, since the X-ray absorption of epoxy resin is low. Thereafter, the same specimen was serially sectioned for LM investigation. The serial section images were aligned and specific organ systems were reconstructed based on manual segmentation and surface rendering. According to the region of interest (ROI), specific LM sections were detached from the slides, re-mounted on resin blocks and re-sectioned (ultrathin) for TEM. For analysis, image data from the three different modalities was co-registered into a single 3D scene using the software AMIRA®. We were able to register both the LM section series volume and TEM slices neatly to the microCT dataset, with small geometric deviations occurring only in the peripheral areas of the specimen. Based on co-registered datasets the excretory organs, which were chosen as ROI for this study, could be investigated regarding both their ultrastructure as well as their position in the organism and their spatial relationship to adjacent tissues. We found structures typical for mollusc excretory systems, including ultrafiltration sites at the pericardial wall, and ducts leading from the pericardium towards the kidneys, which exhibit a typical basal infolding system., Conclusions: The presented approach allows a comprehensive analysis and presentation of small objects regarding both the overall organization as well as cellular and subcellular details. Although our protocol involves a variety of different equipment and procedures, we maintain that it offers savings in both effort and cost. Co-registration of datasets from different imaging modalities can be accomplished with high-end desktop computers and offers new opportunities for understanding and communicating structural relationships within organisms and tissues. In general, the correlative use of different microscopic imaging techniques will continue to become more widespread in morphological and structural research in zoology. Classical TEM serial section investigations are extremely time consuming, and modern methods for 3D analysis of ultrastructure such as SBF-SEM and FIB-SEM are limited to very small volumes for examination. Thus the re-sectioning of LM sections is suitable for speeding up TEM examination substantially, while microCT could become a key-method for complementing ultrastructural examinations.

A single pair of protonephridia is the typical larval excretory organ of molluscs. Their presence in postlarval developmental stages was discovered only recently. We found that the protonephridia of the polyplacophoran mollusc, Lepidochitona corrugata, achieve their most elaborate differentiation and become largest during the postlarval period. This study describes the protonephridia of L. corrugata using light and electron microscopy and interactive three-dimensional visualization. We focus on the postlarval developmental period, in which the protonephridia consist of three parts: the terminal part with the ultrafiltration sites at the distal end, the voluminous protonephridial kidney, and the efferent nephroduct leading to the nephropore. The ultrafiltration sites show filtration slits between regularly arranged thin pedicles. The ciliary flame originates from both the terminal cell and the duct cells of the terminal portion. The efferent duct also shows ciliation. The most conspicuous structures, the protonephridial kidneys, are voluminous swellings composed of reabsorptive cells ("nephrocytes"). These cells exhibit strong vacuolization and an infolding system increasing the basal surface. The protonephridial kidneys, previously not reported at such a level of organization in molluscs, strikingly resemble (metanephridial) kidneys of adult molluscan excretory systems., (Copyright © 2011 Wiley-Liss, Inc.)

We present three examples of interactive, 3D volume rendering models embedded in a PDF publication. The examples are drawn from three different morphological methods - confocal microscopy, serial sectioning and microcomputed tomography - performed on members of the phylum Mollusca. A description of the entire technical procedure from specimen preparation to embedding of the visual model including 3D labels in the document is provided. For comparison, volume rendering with standard visualization software, and surface rendering incorporated in the 3D PDF figures, are provided. The principal advantages and disadvantages of the techniques and models are discussed. Volume rendering for serial sections is relatively work-intensive, while confocal data have limitations in terms of 3D presentation. Volume renderings are normally downsampled in resolution to achieve a reasonable PDF file size, however intentional information is largely retained. We conclude that volume rendering of 3D data sets is a valuable technique and should become standard in PDF versions of biomedical publications.

By providing two examples, the option for embedding 3D models in electronic versions of life science publications is presented. These examples, presumably representing the first such models published, are developmental stages of an evertebrate (Patella caerulea, Mollusca) and a vertebrate species (Psetta maxima, Teleostei) obtained from histological section series reconstruction processed with the software package Amira. These surface rendering models are particularly suitable for a PDF file because they can easily be transformed to a file format required and components may be conveniently combined and hierarchically arranged. All methodological steps starting from specimen preparation until embedding of resulting models in PDF files with emphasis on conversion of Amira data to the appropriate 3D file format are explained. Usability of 3D models in PDF documents is exemplified and advantages over 2D illustrations are discussed, including better explanation capabilities for spatial arrangements, higher information contents, and limiting options for disguising results by authors. Possibilities for additional applications reaching far beyond the examples presented are suggested. Problems such as long-term compatibility of file format and hardware plus software, editing and embedding of files, file size and differences in information contents between printed and electronic version will likely be overcome by technical development and increasing tendency toward electronic at the cost of printed publications. Since 3D visualization plays an increasing role in manifold disciplines of science and appropriate tools for the popular PDF format are readily available, we propose routine application of this way of illustration in electronic life science papers.

Combined electron microscopy and immunocytochemistry of the larvae of several polyplacophoran species (Chiton olivaceus, Lepidochitona aff. corrugata, Mopalia muscosa) revealed a sensory system new to science, a so-called "ampullary system." The cells of the "ampullary system" are arranged in four symmetrically situated pairs lying dorsolaterally and ventrolaterally in the pretrochal part of the trochophore-like larva and they send axons into the cerebral commissure. They are lost at metamorphosis. The fine structure of these cells strongly resembles that of so-called "ampullary cells" known from various sensory organs of other molluscs, such as the apical complex of gastropod and bivalve larvae, osphradia of vetigastropods, and olfactory organs of cephalopods, and nuchal organs of certain polychaetes. The ampullary cells and their nerves are densely stained by anti-FMRF-amide fluorescence dyes, whereas antiserotonin staining is only weak. While cytological homology of the ampullary cells with those of other organs is probable, the ampullary system as a whole is regarded as a synapomorphy of the Polyplacophora or Chitonida., (Copyright 2002 Wiley-Liss, Inc.)

The apical area of larvae of four primitive pulmonate species was investigated by means of serial ultrathin and light microscope sections. Cephalic sensory organs (CSOs) were found in the larvae of Onchidium cf. branchiferum (Onchidiidae) and Laemodonta octanfracta (Ellobiidae), while no trace of the organ was present in the larvae of Ovatella myosotis (Ellobiidae) or Williamia radiata (Siphonariidae). TEM investigation revealed very similar CSOs in O. cf. branchiferum and L. octanfracta, with characteristic putative sensory cell types: ampullary cells with an internal ampulla containing densely packed cilia, para-ampullary cells with external cilia parallel to the surface, and ciliary tuft cells, bearing short ciliary tufts. The epithelium covering the organ has a thick microvillar border with microvilli laterally bearing a pair of electron-dense accumulations and a glycocalyx with interspersed flat plaque-like elements. While homologues of all major elements of the CSO can be found in other gastropod taxa, for example caenogastropods and opisthobranchs, the homology of the ampullary cell with similar cells in nongastropods appears unlikely. The CSO of L. octanfracta is associated with an additional structure, an epithelial external protrusion, lying ventral to the CSO. The absence of the organ in W. radiata weakens hypotheses on the organ's function of examining settlement conditions and velar control., (Copyright 2002 Wiley-Liss, Inc.)