Comparative immunohistochemistry of the cerebral ganglion in Gastrotricha: an analysis of FMRFamide-like immunoreactivity in Neodasys cirritus (Chaetonotida), Xenodasys riedli and Turbanella cf. hyalina (Macrodasyida)

The neuropeptide FMRFamide (Phe–Met–Arg–Phe–NH₂) is part of a large and diverse family of peptidergic neurotransmitters present throughout the animal kingdom. To date, no such neuropeptides have been demonstrated in gastrotrichs despite their presence in closely related invertebrates such as nematodes. Here, the FMRFamidergic nervous system of three marine gastrotrichs is investigated with immunofluorescence, CLSM, and 3D computer imaging to gain insight into structure of the cerebral ganglion and test various phylogenetic hypotheses on its organization. Results reveal that FMRFamide-like immunoreactivity (IR) is present throughout the nervous systems of three species: Neodasys cirritus (Chaetonotida), Xenodasys riedli and Turbanella cf. hyalina (Macrodasyida). Both macrodasyidans possess FMRFamide-like IR in the central, peripheral- and stomatogastric-nervous systems, while FMRFamide-like IR is restricted to the CNS in N. cirritus. In all three species, the cerebral ganglion is dumbbell-shaped and bordered bilaterally by cerebral perikarya: numerous perikarya are present in X. riedli and N. cirritus, while few perikarya are present in T. cf. hyalina. Cerebral perikarya flank the nerve ring neuropil, which contains IR fibers in the supra- and subpharyngeal commissures of both macrodasyidans, but in N. cirritus, only contains IR fibers in the suprapharyngeal commissure. Together, these results confirm the peripharyngeal nature of the gastrotrich cerebral ganglion, but are equivocal on hypotheses of its tripartite structure. Still, the neural organization of gastrotrichs, in particular, the architecture of the cerebral ganglion, is expected to hold valuable information for future assessments of gastrotrich phylogeny, and may yet provide key insights into the evolution of this enigmatic taxon.     

Brain anatomy of the marine tardigrade actinarctus doryphorus (arthrotardigrada)

Knowledge of tardigrade brain structure is important for resolving the phylogenetic relationships of Tardigrada. Here, we present new insight into the morphology of the brain in a marine arthrotardigrade, Actinarctus doryphorus, based on transmission electron microscopy, supported by scanning electron microscopy, conventional light microscopy as well as confocal laser scanning microscopy. Arthrotardigrades contain a large number of plesiomorphic characters and likely represent ancestral tardigrades. They often have segmented body outlines and each trunk segment, with its paired set of legs, may have up to five sensory appendages. Noticeably, the head carries numerous cephalic appendages that are structurally equivalent to the sensory appendages of the trunk segments. Our data reveal that the brain of A. doryphorus is partitioned into three paired lobes, and that these lobes exhibit a more pronounced separation as compared to that of eutardigrades. The first brain lobe in A. doryphorus is located anteriodorsally, with the second lobe just below it in an anterioventral position. Both of these two paired lobes are located anterior to the buccal tube. The third pair of brain lobes are situated posterioventrally to the first two lobes, and flank the buccal tube. In addition, A. doryphorus possesses a subpharyngeal ganglion, which is connected with the first of the four ventral trunk ganglia. The first and second brain lobes in A. doryphorus innervate the clavae and cirri of the head. The innervations of these structures indicate a homology between, respectively, the clavae and cirri of A. doryphorus and the temporalia and papilla cephalica of eutardigrades. The third brain lobes innervate the buccal lamella and the stylets as described for eutardigrades. Collectively, these findings suggest that the head region of extant tardigrades is the result of cephalization of multiple segments. Our results on the brain anatomy of Actinarctus doryphorus support the monophyly of Panarthropoda. J. Morphol. 275:173–190, 2014. © 2013 Wiley Periodicals, Inc.     

Neural Markers Reveal a One-Segmented Head in Tardigrades (Water Bears)

Background

While recent neuroanatomical and gene expression studies have clarified the alignment of cephalic segments in arthropods and onychophorans, the identity of head segments in tardigrades remains controversial. In particular, it is unclear whether the tardigrade head and its enclosed brain comprises one, or several segments, or a non-segmental structure. To clarify this, we applied a variety of histochemical and immunocytochemical markers to specimens of the tardigrade Macrobiotus cf. harmsworthi and the onychophoran Euperipatoides rowelli.

Methodology/Principal Findings

Our immunolabelling against serotonin, FMRFamide and α-tubulin reveals that the tardigrade brain is a dorsal, bilaterally symmetric structure that resembles the brain of onychophorans and arthropods rather than a circumoesophageal ring typical of cycloneuralians (nematodes and allies). A suboesophageal ganglion is clearly lacking. Our data further reveal a hitherto unknown, unpaired stomatogastric ganglion in Macrobiotus cf. harmsworthi, which innervates the ectodermal oesophagus and the endodermal midgut and is associated with the second leg-bearing segment. In contrast, the oesophagus of the onychophoran E. rowelli possesses no immunoreactive neurons, whereas scattered bipolar, serotonin-like immunoreactive cell bodies are found in the midgut wall. Furthermore, our results show that the onychophoran pharynx is innervated by a medullary loop nerve accompanied by monopolar, serotonin-like immunoreactive cell bodies.

Conclusions/Significance

A comparison of the nervous system innervating the foregut and midgut structures in tardigrades and onychophorans to that of arthropods indicates that the stomatogastric ganglion is a potential synapomorphy of Tardigrada and Arthropoda. Its association with the second leg-bearing segment in tardigrades suggests that the second trunk ganglion is a homologue of the arthropod tritocerebrum, whereas the first ganglion corresponds to the deutocerebrum. We therefore conclude that the tardigrade brain consists of a single segmental region corresponding to the arthropod protocerebrum and, accordingly, that the tardigrade head is a non-composite, one-segmented structure.     

Tardigrades — Are They Really Miniaturized Dwarfs?

Tardigrades are animals of small body size which is often regarded to be a secondary phenomenon. This interpretation makes sense in the traditional concept that tardigrades are closely related to Onychophora, Euarthropoda and Annelida. A large body size in the ancestor of this common taxon (Articulata) is probable. Small size and the absence of organs such as a dorsal heart, segmental coelomic cavities and metanephridia must then be interpreted as derived in tardigrades. However, when Cycloneuralia are taken as an outgroup instead of Annelida (taxon Ecdysozoa), an interpretation of small body size as a primary feature is plausible. This also accounts for the absence of heart, coelom and nephridia.The choice of outgroup influences hypotheses about sister-group relationships within Panarthropoda, with either Onychophora (Articulata-concept) or Tardigrada (Ecdysozoa-concept) being basal.     

Patterns of serotonin-immunoreactive neurons in the central nervous system of the earthworm Lumbricus terrestris L.

Summary The distribution patterns of serotonin-immunoreactive somata in the cerebral and subpharyngeal ganglion, and in the head and tail ganglia of the nerve cord of Lumbricus terrestris are described from whole-mount preparations. A small number of serotonin-immunoreactive neurons occurs in the cerebral ganglion, in contrast to the large population of serotonin-immunoreactive neurons that exists in all parts of the ventral nerve cord. From the arrangement of serotonin-immunoreactive somata in the subpharyngeal ganglion, we suggest that this ganglion arises from the fusion of two primordial ganglia. In head and tail ganglia, the distribution of serotonin-immunoreactive somata resembles that in midbody segments. Segmental variations in the pattern and number of serotonin-immunoreactive somata in the different body regions are discussed on the background of known developmental mechanisms that result in metameric neuronal populations in annelids and arthropods.Abbreviations CG1, CG2 cerebral soma group 1, 2 - CNS central nervous system - GINs giant interneurons - 5-HT 5-hydroxytryptamine, serotonin - 5-HTi 5-HT-immunoreactive - N side nerve - SG19 subpharyngeal soma group 1–9 - SN segmental nerve     

Avoidance of host resistance in the oviposition-site preferences of rose bitterling

A contemporary outcome of dynamic host–parasite coevolution can be driven by the adaptation of a parasite to exploit its hosts at the population and species levels (parasite specialisation) or by local host adaptations leading to greater host resistance to sympatric parasite populations (host resistance). We tested the predominance of these two scenarios using cross-infection experiments with two geographically distant populations of the rose bitterling, Rhodeus ocellatus, a fish brood parasite of freshwater mussels, and four populations of their mussel hosts (two Anodonta woodiana and two Unio douglasiae populations) with varying degrees of geographic sympatry and local coexistence. Our data support predictions for host resistance at the species level but no effect of local coexistence between specific populations. Rhodeus ocellatus showed a preference for allopatric host populations, irrespective of host species. Host mussel response, in terms of ejection of R. ocellatus eggs, was stronger in the more widespread and abundant host species (A. woodiana) and this response tended to be higher in sympatric populations. These outcomes provide support for the importance of host resistance in bitterling oviposition-site decisions, demonstrating that host choice by R. ocellatus is adaptive by minimizing egg ejections. These findings imply that R. ocellatus, and potentially other bitterling species, may benefit from exploiting novel hosts, which may not possess appropriate adaptive responses to parasitism.     

Anatomy of neurons crossing the tritocerebral commissures of the cockroach Periplaneta americana (Blattaria)

The neuronal connections of the tritocerebral commissures of Periplaneta americana were studied in the brain-suboesophageal ganglion complex and the stomatogastric nervous system by means of heavy metal iontophoresis through cut nerve ends followed by silver intensification. The tritocerebral commissure 1 (Tc1) contains mainly the processes of the subpharyngeal nerve (Spn) whose neurons are located in both tritocerebral lobes and in the frontal ganglion. Some neurons of the frontal ganglion project through the Tc1 to the contralateral tritocerebrum. A few fibers in this commissure were observed projecting to the protocerebrum and the suboesophageal ganglion. There are tritocerebral neurons which pass through the Tc1 or the tritocerebral commissure 2 (Tc2) and extend on into the stomatogastric nervous system. One axon of a descending gaint neuron appears in the Tc2. This neuron lies in the tritocerebrum and connects the brain to the contralateral side of the ventral nerve cord. In addition, sensory fibers of the labral nerve (Ln) traverse both commissures to the opposite tritocerebrum. The anatomical and physiological relevance of the identified neuronal pathways is discussed. © 1995 Wiley-Liss, Inc.     

What remains after 2 months of starvation? Analysis of sequestered algae in a photosynthetic slug, Plakobranchus ocellatus (Sacoglossa, Opisthobranchia), by barcoding

The sacoglossan sea slug, Plakobranchus ocellatus, is a so-called long-term retention form that incorporates chloroplasts for several months and thus is able to starve while maintaining photosynthetic activity. Little is known regarding the taxonomy and food sources of this sacoglossan, but it is suggested that P. ocellatus is a species complex and feeds on a broad variety of Ulvophyceae. In particular, we analysed specimens from the Philippines and starved them under various light conditions (high light, low light and darkness) and identified the species of algal food sources depending on starvation time and light treatment by means of DNA-barcoding using for the first time the combination of two algal chloroplast markers, rbcL and tufA. Comparison of available CO1 and 16S sequences of specimens from various localities indicate a species complex with likely four distinct clades, but food analyses do not indicate an ecological separation of the investigated clades into differing foraging strategies. The combined results from both algal markers suggest that, in general, P. ocellatus has a broad food spectrum, including members of the genera Halimeda, Caulerpa, Udotea, Acetabularia and further unidentified algae, with an emphasis on H. macroloba. Independent of the duration of starvation and light exposure, this algal species and a further unidentified Halimeda species seem to be the main food source of P. ocellatus from the Philippines. It is shown here that at least two (or possibly three) barcode markers are required to cover the entire food spectrum in future analyses of Sacoglossa.     

Nine viruses from eight lineages exhibiting new evolutionary modes that co-infect a hypovirulent phytopathogenic fungus

Mycoviruses are an important component of the virosphere, but our current knowledge of their genome organization diversity and evolution remains rudimentary. In this study, the mycovirus composition in a hypovirulent strain of Sclerotinia sclerotiorum was molecularly characterized. Nine mycoviruses were identified and assigned into eight potential families. Of them, six were close relatives of known mycoviruses, while the other three had unique genome organizations and evolutionary positions. A deltaflexivirus with a tripartite genome has evolved via arrangement and horizontal gene transfer events, which could be an evolutionary connection from unsegmented to segmented RNA viruses. Two mycoviruses had acquired a second helicase gene by two different evolutionary mechanisms. A rhabdovirus representing an independent viral evolutionary branch was the first to be confirmed to occur naturally in fungi. The major hypovirulence-associated factor, an endornavirus, was finally corroborated. Our study expands the diversity of mycoviruses and potential virocontrol agents, and also provides new insights into virus evolutionary modes including virus genome segmentation.     

Armadillo-like helical domain containing-4 is dynamically expressed in both the first and second heart fields

Armadillo repeat and Armadillo-like helical domain containing proteins form a large family with diverse and fundamental functions in many eukaryotes. Herein we investigated the spatiotemporal expression pattern of Armadillo-like helical domain containing 4 (or Armh4) as an uncharacterized protein coding mouse gene, within the mouse embryo during the initial stages of heart morphogenesis. We found Armh4 is initially expressed in both first heart field as well as the second heart field progenitors and subsequently within predominantly their cardiomyocyte derivatives. Armh4 expression is initially cardiac-restricted in the developing embryo and is expressed in second heart field subpharyngeal mesoderm prior to cardiomyocyte differentiation, but Armh4 diminishes as the embryonic heart matures into the fetal heart. Armh4 is subsequently expressed in craniofacial structures and neural crest-derived dorsal root and trigeminal ganglia. Whereas lithium chloride-induced stimulation of Wnt/β-catenin signaling elevated Armh4 expression in both second heart field subpharyngeal mesodermal progenitors and outflow tract, right ventricle and atrial cardiomyocytes, neither a systemic loss of Islet-1 nor an absence of cardiac neural crest cells had any effect upon Armh4 expression. These results confirm that Wnt/β-catenin-responsive Armh4 is a useful specific biomarker of the FHF and SHF cardiomyocyte derivatives only.     

Caste and sex specific olfactory glomerular organization and brain architecture in two sympatric ant species Camponotus sericeus and Camponotus compressus (Fabricius, 1798)

We use monoclonal antibodies against synaptic proteins and anterograde tracing with neurobiotin to describe the architecture of the antennal lobes in different castes of two ant species – Camponotus sericeus and Camponotus compressus. The reproductives and worker classes are readily categorized based on size and external morphology. The overall organization of brain neuropile is comparable between castes with differences only in the visual ganglia. Males have a larger fraction of neuropile occupied by the medulla and lobula than females. In the diurnal species, C. sericeus these regions are more highly represented, than in the nocturnal species C. compressus. The most striking differences are in the antennal lobe where males possess a macroglomerulus, which is about ten times larger in volume than the other glomeruli; such a specialization is absent in females. Minor workers possess a significantly larger number of glomeruli than the majors despite the smaller overall volume of the lobe. These caste-specific differences occur mainly within glomerular clusters that receive input from sensory neurons that project in tracts – T4 and T5 – within the antennal nerve. The comparative anatomy of different castes of ants provides an entry point into a future systematic analysis of how divergent brain architectures can arise within a single species.     

Parasegmental appendage allocation in annelids and arthropods and the homology of parapodia and arthropodia

The new animal phylogeny disrupts the traditional taxon Articulata (uniting arthropods and annelids) and thus calls into question the homology of the body segments and appendages in the two groups. Recent work in the annelid Platynereis dumerilii has shown that although the set of genes involved in body segmentation is similar in the two groups, the body units of annelids correspond to arthropod parasegments not segments. This challenges traditional ideas about the homology of "segmental" organs in annelids and arthropods, including their appendages. Here I use the expression of engrailed, wingless and Distal-less in the arthropod Artemia franciscana to identify the parasegment boundary and the appendage primordia. I show that the early body organization including the appendage primordia is parasegmental and thus identical to the annelid organization and by deriving the different adult appendages from a common ground plan I suggest that annelid and arthropod appendages are homologous structures despite their different positions in the adult animals. This also has implications for the new animal phylogeny, because it suggests that Urprotostomia was not only parasegmented but also had parasegmental appendages similar to extant annelids, and that limb-less forms in the Protostomia are derived from limb-bearing forms.     

Structure of reconstituted bacterial membrane efflux pump by cryo-electron tomography

Complexes of OprM and MexA, two proteins of the MexA-MexB-OprM multidrug efflux pump from Pseudomonasaeruginosa, an opportunistic Gram-negative bacterium, were reconstituted into proteoliposomes by detergent removal. Stacks of protein layers with a constant height of 21 nm, separated by lipid bilayers, were obtained at stoichiometry of 1:1 (w/w). Using cryo-electron microscopy and tomography, we showed that these protein layers were composed of MexA-OprM complexes self-assembled into regular arrays. Image processing of extracted sub-tomograms depicted the architecture of the bipartite complex sandwiched between two lipid bilayers, representing an environment close to that of the native whole pump (i.e. anchored between outer and inner membranes of P. aeruginosa). The MexA-OprM complex appeared as a cylindrical structure in which we were able to identify the OprM molecule and the MexA moiety. MexA molecules have a cylindrical shape prolonging the periplasmic helices of OprM, and widening near the lipid bilayer. The flared part is likely composed of two MexA domains adjacent to the lipid bilayer, although their precise organization was not reachable mainly due to their flexibility. Moreover, the intermembrane distance of 21 nm indicated that the height of the bipartite complex is larger than that of the tripartite AcrA-AcrB-TolC built-up model in which TolC and AcrB are docked into contact. We proposed a model of MexA-OprM taking into account features of previous models based on AcrA-AcrB-TolC and our structural results providing clues to a possible mechanism of tripartite system assembly.     

The brain of Echiniscus viridissimus Peterfi, 1956 (Heterotardigrada): a key to understanding the phylogenetic position of tardigrades and the evolution of the arthropod head

The brain of Echiniscus viridissimus , Peterfi, 1956 is composed of a series of orthogonally arranged neuropils. The most anterior neuropils are rireumbuccal, positioned dorso-and ventrolateral to the buccal tube and are associated with ganglia for sensory receptors of the mouth cone. Posterior to these are neuropils and ganglia for the (1) internal cirri and (2) cephalic papillae, external cirri, cirri A and clavae. They are joined by two pairs of vertical tracts to neuropils lateral to the buccal tube. A model based on the postcephalic organization of the tardigrade nervous system is used to propose a transformation of segmental ganglia that gives an arrangement congruent with the pattern of neuropils in the brain. The analysis suggests that the brain is derived from nervous elements of four segments with the fourth segment having contributed paired dorsal ganglia and their connecting vertical tracts to the first trunk ganglia of the ventral chain. The organization of the head of tardigrades is compared with that of other lobopods and arthropods and several possible key evolutionary innovations are offered. In addition homologous characters for the heads of tardigrades and other lobopods and arthropods are proposed and the nomenclature for the tardigrade cephalic nervous system is discussed.     

Venom proteomic analysis of medically important Nigerian viper Echis ocellatus and Bitis arietans snake species

Snakebite envenoming remains a neglected tropical disease which poses severe health hazard, especially for the rural inhabitants in Africa. In Nigeria, vipers are responsible for the highest number of deaths. Hydrophilic interaction liquid chromatography coupled with LC-MS/MS was used to analyze the crude venoms of Echis ocellatus (Carpet viper) and Bitis arietans (Puff adder) in order to understand their venom proteomic identities. Results obtained revealed that gel-free proteomic analysis of the crude venoms led to the identification of 85 and 79 proteins, respectively. Seventy-eight (78) proteins were common between the two snake species with a 91.8% similarity score. The identified proteins belong to 18 protein families in E. ocellatus and 14 protein families in B. arietans. Serine proteases (22.31%) and metalloproteinases (21.06%) were the dominant proteins in the venom of B. arietans; while metalloproteinases (34.84%), phospholipase A₂s (21.19%) and serine proteases (15.50%) represent the major toxins in the E. ocellatus venom. Other protein families such as three-finger toxins and cysteine-rich venom proteins were detected in low proportions. This study provides an insight into the venom proteomic analysis of the two Nigerian viper species, which could be useful in identifying the toxin families to be neutralized in case of envenomation.