Ultrastructure and histochemistry of the male reproductive system of the genus Callinectes Stimpson, 1860 (Brachyura: Portunidae).

We described the ultrastructure and histochemistry of the reproductive system of five Callinectes species, and evaluate the seasonal variation in weight of the reproductive system and hepatopancreas by comparing annual changes of somatic indices. The somatic indices changed little throughout the year. In Callinectes, spermatogenesis occurs inside the lobular testes and, within each lobule, the cells are at the same developmental stage. Spermatogenesis and spermiogenesis follow the same development pattern in all Callinectes studied. Mature spermatozoa are released into the seminiferous ducts through the collecting ducts. Cells of the vas deferens are secretory as evidenced by rough endoplasmic reticulum, Golgi complex, and secretory vesicles that produce the seminal fluid. The anterior vas deferens shows two portions: proximal and distal. In proximal portion (AVDp), spermatozoa are clustered and embedded in an electron-dense, basophilic glycoproteinaceous secretion Type I. In the distal portion (AVDd), the spermatophore wall is formed by incorporation of a less electron-dense glycoproteinaceous secretion Type II. The secretion Type I change to an acid polysaccharide-rich matrix that separates the spermatophores from each other. The median vas deferens (MVD) stores the spermatophores and produces the granular glycoproteinaceous seminal fluid. The posterior vas deferens (PVD) has few spermatophores. Its epithelium has many mitochondria and the PVD seminal fluid changes into a liquid and homogeneous glycoprotein. Many outpocketings in the PVD and MVD help to increase the fluid production. Overall, the reproductive pattern of Callinectes is similar to other species that produce sperm plugs. The secretions of AVD, MVD, and PVD are responsible for the polymerization that forms the solid, waxy plug in the seminal receptacle. The traits identified here are common to all Portunidae species studied so far.

Direction Selective Neurons Responsive to Horizontal Motion in a Crab Reflect an Adaptation to Prevailing Movements in Flat Environments.

All animals need information about the direction of motion to be able to track the trajectory of a target (prey, predator, cospecific) or to control the course of navigation. This information is provided by direction selective (DS) neurons, which respond to images moving in a unique direction. DS neurons have been described in numerous species including many arthropods. In these animals, the majority of the studies have focused on DS neurons dedicated to processing the optic flow generated during navigation. In contrast, only a few studies were performed on DS neurons related to object motion processing. The crab Neohelice is an established experimental model for the study of neurons involved in visually-guided behaviors. Here, we describe in male crabs of this species a new group of DS neurons that are highly directionally selective to moving objects. The neurons were physiologically and morphologically characterized by intracellular recording and staining in the optic lobe of intact animals. Because of their arborization in the lobula complex, we called these cells lobula complex directional cells (LCDCs). LCDCs also arborize in a previously undescribed small neuropil of the lateral protocerebrum. LCDCs are responsive only to horizontal motion. This nicely fits in the behavioral adaptations of a crab inhabiting a flat, densely crowded environment, where most object motions are generated by neighboring crabs moving along the horizontal plane.SIGNIFICANCE STATEMENT Direction selective (DS) neurons are key to a variety of visual behaviors including, target tracking (preys, predators, cospecifics) and course control. Here, we describe the physiology and morphology of a new group of remarkably directional neurons exclusively responsive to horizontal motion in crabs. These neurons arborize in the lobula complex and in a previously undescribed small neuropil of the lateral protocerebrum. The strong sensitivity of these cells for horizontal motion represents a clear example of functional neuronal adaptation to the lifestyle of an animal inhabiting a flat environment.

Neuronal morphologies built for reliable physiology in a rhythmic motor circuit.

It is often assumed that highly-branched neuronal structures perform compartmentalized computations. However, previously we showed that the Gastric Mill (GM) neuron in the crustacean stomatogastric ganglion (STG) operates like a single electrotonic compartment, despite having thousands of branch points and total cable length >10 mm (Otopalik et al., 2017a; 2017b). Here we show that compact electrotonic architecture is generalizable to other STG neuron types, and that these neurons present direction-insensitive, linear voltage integration, suggesting they pool synaptic inputs across their neuronal structures. We also show, using simulations of 720 cable models spanning a broad range of geometries and passive properties, that compact electrotonus, linear integration, and directional insensitivity in STG neurons arise from their neurite geometries (diameters tapering from 10-20 µm to < 2 µm at their terminal tips). A broad parameter search reveals multiple morphological and biophysical solutions for achieving different degrees of passive electrotonic decrement and computational strategies in the absence of active properties.

Histological analysis of oogenesis and ovarian development of the freshwater crab Sinopotamon henanense.

The morphological and cytological changes of oogenesis and ovarian development were described in the freshwater crab Sinopotamon henanense through macroscopic and microscopic examinations. Serial histological dissections of the ovaries demonstrated that oocyte development was asynchronous. Oogenesis was divided into four distinct stages including six phases: oogonium stage, the first phase (OI) and the second phase (OII) comprising the previtellogenic stage, the third phase (OIII), the fourth phase (OIV) and the fifth phase (OV), comprising the vitellogenic stage and the sixth phase representing the mature stage. Furthermore, examining and analyzing the gonadosomatic indices showed that the developmental cycle of the ovary was closely related to season, and indicated that the breeding season of S. henanense was between May and June. Ovarian development was classified into six stages: proliferation stage, small growth stage, large growth stage, pre-maturation stage, mature stage and spawning stage. Ovaries varied in size and color during each developmental stage, which were closely related to the status and proportions of oogonia and primary oocytes. Although there were cases that oocytes at two or more phases were present at each stage, ovary developmental stages were substantially different. These results provide an important base for studies of the regulatory mechanisms of oogenesis in this compared to other brachyuran species, and will be useful for the aquaculture of S. henanense and related species.

Oxidative stress and genotoxic effect of deltamethrin exposure on the Chinese mitten crab, Eriocheir sinensis.

The oxidative stress and genotoxic effect of deltamethrin on the Chinese mitten crab Eriocheir sinensis were assessed using several commonly used biomarkers in this study. The results showed that the 48 h and 96 h LC50 values of deltamethrin to E. sinensis were 2.319 and 1.164 µg/L, respectively, and the safe concentration was 0.293 µg/L. According to these results, deltamethrin was applied at concentrations of 1/16, 1/8, 1/4, 1/2 and 1/1 96 h LC50 for 8 d in an exposure experiment. The activity of superoxide dismutase (SOD) increased remarkably at 1 d, but decreased at 4 d in concentration group of 1/4, 1/2 and 1/1 LC50, whereas catalase (CAT) activity decreased during the exposure. The total antioxidant capacity (T-AOC) at the concentrations of 1/4, 1/8 and 1/16 LC50 increased significantly at 1 d or 2 d respectively, whereas it decreased gradually until the end of the experiment under the concentrations above 1/4 LC50. The oxidative stress products malondialdehyde (MDA) and hydrogen peroxide (H2O2) in serum increased significantly at each concentration except H2O2 at concentration of 1/16 LC50. Additionally, the micronucleus (MN) frequency of haemocytes increased at the concentrations of 1/4, 1/2 and 1/1 LC50 throughout the exposure, similar trend was observed in the comet ratio and percentage of tail-DNA (%DNA) in haemocytes. These results revealed that deltamethrin had a prominent toxic effect on E. sinensis based on antioxidative response inhibition and genotoxicity that was possibly due to lipid peroxidation (LPO) induced by oxidative products, and the accumulation of peroxide, even under a sublethal concentration.

Transcriptomic analysis of crustacean molting gland (Y-organ) regulation via the mTOR signaling pathway.

The intermolt crustacean Y-organ (YO) maintains a basal state mediated by pulsatile release of molt inhibiting hormone (MIH), a neuropeptide produced in the eyestalk ganglia, inhibiting YO ecdysteroidogenesis. Reduction of MIH results in YO activation and the animal enters premolt. In the crab, Gecarcinus lateralis, molting was induced by eyestalk ablation (ESA). ESA animals were injected with either rapamycin, an mTOR inhibitor, or DMSO vehicle at Day 0. YOs were harvested at 1, 3, and 7 days post-ESA and processed for high throughput RNA sequencing. ESA-induced increases in mRNA levels of mTOR signaling genes (e.g., mTOR, Rheb, TSC1/2, Raptor, Akt, and S6 kinase) declined following rapamycin treatment. In concert with mTOR inhibition, mRNA levels of ecdysteroid biosynthesis genes (e.g., Nvd, Spo, Sad, Dib, and Phm) were decreased and accompanied by a decrease in hemolymph ecdysteroid titer. By contrast, rapamycin increased the mRNA level of FKBP12, the rapamycin-binding protein, as well as the mRNA levels of genes associated with Wnt and insulin-like growth factor signaling pathways. Many MIH and transforming growth factor-ß signaling genes were down regulated in ESA animals. These results indicate that mTOR activity either directly or indirectly controls transcription of genes that drive activation of the YO.

Does the bone morphogenetic protein 7 inhibit oocyte maturation by autocrine/paracrine in mud crab?

A bone morphogenetic protein ligand (BMP7) and its two receptors (BMPRIB and BMPRII) were recently cloned and characterized in the mud crab, Scylla paramamosain. However specific functions of BMP7 and the mechanistic pathways regulating its function are largely unidentified. In the present study, we separated oocytes and follicle cells from the ovarian explants of S. paramamosain. Subsequent analysis using semi-quantitative PCR demonstrated that the mRNA of Sp-BMP7 was exclusively expressed in follicle cells while Sp-BMPRs were expressed in both oocytes and follicle cells. In vitro experiments further showed that the mRNA and protein levels of Cyclin B increased but Sp-BMP7 declined in 17α, 20ß-Dihydroxyprogesterone (DHP)-induced oocytes. Furthermore, the inhibitory effects of Sp-BMP7 were not affected by the elimination of the contact/gap junction-mediated communication between oocytes and follicle cells. Our data indicate that BMP7 may play a role in the suppression of DHP-induced oocyte maturation by affecting autocrine/paracrine pathways in S. paramamosain.

Hemocytes of the mud crab Scylla paramamosain: Cytometric, morphological characterization and involvement in immune responses.

Hemocytes play essential roles in the innate immune system of crustaceans. Characterization of hemocytes from estuary mud crab Scylla paramamosain was performed by flow cytometry and morphological studies such as cytochemical staining and electron microscopy. The hemocyte subsets were further separated using a modified Percoll density gradient centrifugation method. Based on the morphological characteristics of the cells, three distinct categories of hemocytes were identified: granulocytes with abundant large granularity representing 5.27 ± 0.42%, semigranulocytes with small or less granularity representing 76.03 ± 3.34%, and hyalinocytes (18.70 ± 3.92%) which were almost no granularity. The total hemocyte cell count and the percentage of hemocyte subsets varied after pathogen infection, including Vibrio alginolyticus and the viral double-stranded RNA analog Poly (I:C). The phagocytic process is of fundamental importance for crustaceans' cellular immune response as well as development and survival. The results of the in vitro phagocytosis assays analyzed by flow cytometry demonstrated that granulocytes and semigranulocytes had significantly higher phagocytic ability than hyalinocytes. A primary culture system, L-15 medium supplemented with 5-10% fetal bovine serum, was developed to further investigate the immune function of hemocytes. Furthermore, adenovirus can be utilized to effectively transfer GFP gene into hemocytes. Overall, three hemocyte sub-populations of S. paramamosain were successfully discriminated, moreover, their response to pathogen infections, phagocytic activity and adenovirus mediated transfection were also investigated for the first time. This study may contribute to a better understanding of the innate immune system of estuary crabs.

Functional characterization of hemocytes from Chinese mitten crab Eriocheir sinensis by flow cytometry.

Hemocytes comprise a diversity of cell types with functional and structural heterogeneity, and they play key roles in the host defense of invertebrates. In the present study, the hemocytes from Chinese mitten crab Eriocheir sinensis were directly separated into two groups by flow cytometry. The hemocytes in P1 group were full of round and abundant granules with deeply staining cytoplasm, while P2 hemocytes were more diverse with a wide range of sizes and less granularity. Both P1 and P2 hemocytes exhibited phagocytic ability, but the phagocytic rate of P1 hemocytes increased which was significantly higher than that of P2 hemocytes after LPS stimulations. The levels of ROS production and intracellular Calcium as well as lysosome content were higher in P1 hemocytes than that in P2 hemocytes under both normal and immune-activated situations. The genes involved in phagocytosis, antimicrobial and antioxidant activities were mainly expressed in P1 hemocytes, while the genes involved in proPO activation system were highly expressed in P2 hemocytes. These results collectively suggested that P1 hemocytes were the main immunocompetent hemocytes in Chinese mitten crab and P2 hemocytes mainly participated in proPO activation system.

Hemocytes and hemocytic responses in the mole crab Emerita emeritus (Linnaeus 1767).

The mole crab, Emerita emeritus, collected from the sandy shores of a Chennai beach, was investigated for cellular immune responses based on the morphology and defensive reactions of the circulating haemocytes. Three haemocyte morphotypes were identified using light and electron microscopy, and separated in a discontinuous percoll gradient. A phagocytosis study using human B erythrocyte as a target cell under phase-contrast optics showed that granular and semi-granular haemocytes were phagocytic, and this response was enhanced by using serum (opsonin)-coated human B erythrocyte in unfractionated and fractionated haemocytes. Observation of TEM image of phagocytosis revealed that the initial recognition and binding of the target cell was restricted to granular and semigranular haemocytes, which were lacking with hyaline cells. However, the encapsulation of DEAE Sepharose CL 6B beads, either untreated or coated with serum (opsonin), was restricted to hyaline cells. This suggests the occurrence of two cell lines in haemocytes, based on the differences observed in the response of haemocytes to bind target cells for phagocytosis or encapsulation. The present study also differentiated the activation of PO in the plasma, serum, and haemocyte lysate supernatant (HLS).

Hepatopancreatic cells of a stone crab Menippe frontalis from Perú: separation, viability study, and evaluation of lipoperoxidation against cadmium contamination.

Crustaceans are frequently used as bioindicators, and changes in their metabolism at the hepatopancreas (HP) level are often followed in these studies. The HP is the site of digestion, absorption, nutrient storage, and toxic metal detoxification, enabling crabs to survive in metal contaminated regions. Cellular damage and high lipid peroxidation (LPO) levels have been found in crab populations under high cadmium (Cd) concentrations. The aim of this work was to separate and characterize the HP cells of the stone crab Menippe frontalis from the Pacific Ocean, Perú (5° 5' 21″ S-81° 6' 51″ W) and to measure the cellular viability and LPO after exposure to the non-essential metal Cd. The HP cells were dissociated by magnetic stirring, with posterior separation by sucrose gradient at concentrations of 10, 20, 30, and 40%. We found the same cell types that were described for other species (e.g., Ucides cordatus, Atlantic Ocean, Brazil). High cellular viability against 1 mmol L-1 of Cd was observed for resorptive (R) cells in 20% sucrose layer (88 ± 8%, *P < 0.05, ANOVA), and blister (B) cells in the 40% sucrose layers (92 ± 7%, *P < 0.05, ANOVA). Cd (1 mmol L-1) caused an increase in LPO levels, suggesting that crabs from polluted areas can be affected by toxic metals, generating a physiological stress. The gradient sucrose methodology can be used for different species and results in a similar separation, viability, and cellular identification. The results are a starting point for toxic metal studies for species distributed across different geographic coordinates.

Mechanisms of generation of membrane potential resonance in a neuron with multiple resonant ionic currents.

Neuronal membrane potential resonance (MPR) is associated with subthreshold and network oscillations. A number of voltage-gated ionic currents can contribute to the generation or amplification of MPR, but how the interaction of these currents with linear currents contributes to MPR is not well understood. We explored this in the pacemaker PD neurons of the crab pyloric network. The PD neuron MPR is sensitive to blockers of H- (IH) and calcium-currents (ICa). We used the impedance profile of the biological PD neuron, measured in voltage clamp, to constrain parameter values of a conductance-based model using a genetic algorithm and obtained many optimal parameter combinations. Unlike most cases of MPR, in these optimal models, the values of resonant- (fres) and phasonant- (fϕ = 0) frequencies were almost identical. Taking advantage of this fact, we linked the peak phase of ionic currents to their amplitude, in order to provide a mechanistic explanation the dependence of MPR on the ICa gating variable time constants. Additionally, we found that distinct pairwise correlations between ICa parameters contributed to the maintenance of fres and resonance power (QZ). Measurements of the PD neuron MPR at more hyperpolarized voltages resulted in a reduction of fres but no change in QZ. Constraining the optimal models using these data unmasked a positive correlation between the maximal conductances of IH and ICa. Thus, although IH is not necessary for MPR in this neuron type, it contributes indirectly by constraining the parameters of ICa.

Muscles innervated by a single motor neuron exhibit divergent synaptic properties on multiple time scales.

Adaptive changes in the output of neural circuits underlying rhythmic behaviors are relayed to muscles via motor neuron activity. Presynaptic and postsynaptic properties of neuromuscular junctions can impact the transformation from motor neuron activity to muscle response. Further, synaptic plasticity occurring on the time scale of inter-spike intervals can differ between multiple muscles innervated by the same motor neuron. In rhythmic behaviors, motor neuron bursts can elicit additional synaptic plasticity. However, it is unknown whether plasticity regulated by the longer time scale of inter-burst intervals also differs between synapses from the same neuron, and whether any such distinctions occur across a physiological activity range. To address these issues, we measured electrical responses in muscles innervated by a chewing circuit neuron, the lateral gastric (LG) motor neuron, in a well-characterized small motor system, the stomatogastric nervous system (STNS) of the Jonah crab, Cancer borealisIn vitro and in vivo, sensory, hormonal and modulatory inputs elicit LG bursting consisting of inter-spike intervals of 50-250 ms and inter-burst intervals of 2-24 s. Muscles expressed similar facilitation measured with paired stimuli except at the shortest inter-spike interval. However, distinct decay time constants resulted in differences in temporal summation. In response to bursting activity, augmentation occurred to different extents and saturated at different inter-burst intervals. Further, augmentation interacted with facilitation, resulting in distinct intra-burst facilitation between muscles. Thus, responses of multiple target muscles diverge across a physiological activity range as a result of distinct synaptic properties sensitive to multiple time scales.

Comparative study of the morphology of the female seminal receptacles of Ilia nucleus and Persephona mediterranea (Decapoda, Brachyura, Leucosiidae).

Because of the poor knowledge of the morphology of the female reproductive organs of most brachyuran crabs, this study investigated two Atlantic representatives of the family Leucosiidae, Ilia nucleus (Linnaeus, 1758) and Persephona mediterranea (Herbst, 1794), using histological methods and magnetic resonance imaging (MRI). While the vagina conforms to the concave type, the arrangement of the two chambers of the seminal receptacle differs strongly from that of other eubrachyuran sperm storage organs. Both chambers are oriented laterally within the crab's body. This is in contrast to the dorso-ventral orientation described in most other known brachyuran crabs. The lateral chamber is covered by cuticle, whereas the medial chamber is covered by a holocrine glandular epithelium. The oviduct connection is located ventrally, posterior to the vagina. The oviduct orifice is characterized by a transition from the epithelium lining the oviduct to the seminal receptacle's holocrine glandular epithelium. Moreover, muscle fibres are attached to the oviduct orifice and to the sternal cuticle. This musculature can be interpreted as an important feature in the fertilization and egg-laying process by supporting and controlling the inflow of eggs into the seminal receptacle lumen. The results of this study are compared to the morphology of the seminal receptacle of another leucosiid crab, Ebalia tumefacta (Montagu, 1808), and to those of other known eubrachyuran crabs.

Gliocyte and synapse analyses in cerebral ganglia of the Chinese mitten crab, Eriocheir sinensis: ultrastructural study.

The Chinese mitten crab Eriocheir sinensis is an economically important aquatic species in China. Many studies on gene structure, breeding, and diseases of the crab have been reported. However, knowledge about the organization of the nerve system of the crab remains largely unknown. To study the ultrastructure of the cerebral ganglia of E. sinensis and to compare the histological findings regarding the nerve systems of crustaceans, the cerebral ganglia were observed by transmission electron microscopy. The results showed that four types of gliocytes, including type I, II, III, and IV gliocytes were located in the cerebral ganglia. In addition, three types of synapses were present in the cerebral ganglia, including unidirectional synapses, bidirectional synapses, and combined type synapses.

Localization and identification of crustacean hyperglycemic hormone producing neurosecretory cells in the eyestalk of blue swimmer crab, Portunus pelagicus.

This study intensely focuses on to the localization and identification of crustacean hyperglycemic hormone (CHH) producing neurosecretory cells in the eyestalk of the blue swimmer crab Portunus pelagicus. Anti-Carcinus maenas-CHH was used to identify the location of CHH neurosecretory cells by immunohistochemistry. Ten pairs of eyestalks were collected from intact adult intermoult female crab and fixed in Bouin's fixative. Eyestalks were serially sectioned and stained with chrome-hematoxylin-phloxine stain. Histological studies show the presence of different types of neurosecretory cells namely A (multipolar), B (tripolar), C (bipolar), D (unipolar), E (oval), and F (spherical) in the medulla interna, externa, and terminalis regions based on their size, shape, and tinctorial properties. The neurohemal organ, sinus gland (SG) was observed laterally between medulla interna and terminalis regions. Immunohistochemical studies showed the presence of distinct CHH-like immunoreactivity in the optic ganglia. Divergent group of neurosecretory cells with varying degree of immunoreactivity with Anti-Carcinus maenas-CHH (low, moderate, and intense reactivity) were identified in medulla terminalis, medulla interna, medulla externa, and sinus gland. The present study maps the various types of neurosecretory cells in the optic ganglia and also shows the presence of CHH-like immunoreactivity in various regions of optic ganglia in P. pelagicus. The presence of these unique neurosecretory cell types with larger cell diameter in medulla terminalis, a region that bears the neurosecretory cell bodies, suggest high secretory activity.

The characterization of hematopoietic tissue in adult Chinese mitten crab Eriocheir sinensis.

Invertebrates rely on the efficient innate immune mechanisms against invaders, in which the continuous production of hemocytes (hematopoiesis) is indispensable. In the present study, the hematopoietic tissue (HPT) from Chinese mitten crab Eriocheir sinensis was identified and characterized. It was a thin and non-transparent sheet located at the dorsolateral side of the stomach, which was composed of a series of ovoid lobules. Each lobule was surrounded by connective tissue containing a large amount of spherical cells with big nucleus. In HPT, the cells were full of mitochondria and granules, and DNA replication was detected in some cells by EdU labeling technique. Cell proliferation was observed in HPT by transmission electron microscope (TEM). The distribution of two transcription factors, GATA1 and RUNX1, were examined by human GATA1 and RUNX1 antibodies, respectively. Three homologues of RUNX1 were detected in the HPT while no signal of RUNX1 was observed in hemocytes, and GATA1 was detected in both HPT and some hemocytes. The mRNA transcript of a novel hematopoiesis related cytokine EsAst was detected in hepatopancreas and hemocytes, but it was no detectable in HPT. The mRNA expression level of EsAst in hepatopancreas was 1.38-fold higher than that in hemocytes. Total hemocytes counts were related to the mRNA expression level of EsAst post Aeromonas hydrophila challenge. The results suggested that the stem cells in the hematopoietic tissue of Chinese mitten crab E. sinensis were regulated by transcriptional and humoral factors to generate hemocytes.

Cadmium Induces Apoptosis in Freshwater Crab Sinopotamon henanense through Activating Calcium Signal Transduction Pathway.

Calcium ion (Ca2+) is one of the key intracellular signals, which is implicated in the regulation of cell functions such as impregnation, cell proliferation, differentiation and death. Cadmium (Cd) is a toxic environmental pollutant that can disturb cell functions and even lead to cell death. Recently, we have found that Cd induced apoptosis in gill cells of the freshwater crab Sinopotamon henanense via caspase activation. In the present study, we further investigated the role of calcium signaling in the Cd-induced apoptosis in the animals. Our data showed that Cd triggered gill cell apoptosis which is evidenced by apoptotic DNA fragmentation, activations of caspases-3, -8 and -9 and the presence of apoptotic morphological features. Moreover, Cd elevated the intracellular concentration of Ca2+, the protein concentration of calmodulin (CaM) and the activity of Ca2+-ATPase in the gill cells of the crabs. Pretreatment of the animals with ethylene glycol-bis-(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), Ca2+ chelator, inhibited Cd-induced activation of caspases-3, -8 and -9 as well as blocked the Cd-triggered apoptotic DNA fragmentation. The apoptotic morphological features were no longer observed in gill cells pretreated with the Ca2+ signaling inhibitors before Cd treatment. Our results indicate that Cd evokes gill cell apoptosis through activating Ca2+-CaM signaling transduction pathway.

The branchiostegal lung of Uca vocans (Decapoda: Ocypodidae): Unreported complexity revealed by corrosion casting and MicroCT techniques.

The study of adaptation to terrestrial life in crabs poses several physiological questions. One of the major challenges the crabs have to face is respiration of air: most of the time, gills are unsuitable to perform oxygen exchange out of the water. Fiddler crabs, like other representatives of the Ocypodidae, have developed an additional mechanism of respiration by improving the circulation that lines the branchiostegal chamber, thus developing a branchiostegal lung. In the present study we describe the hitherto unreported complex morphology of the branchiostegal lung of the fiddler crab Uca vocans by means of corrosion casting techniques and 3D reconstruction. This complexity leads us to reconsider the degree of terrestriality of U. vocans and its evolutionary pathway towards land.

A network of visual motion-sensitive neurons for computing object position in an arthropod.

Highly active insects and crabs depend on visual motion information for detecting and tracking mates, prey, or predators, for which they require directional control systems containing internal maps of visual space. A neural map formed by large, motion-sensitive neurons implicated in processing panoramic flow is known to exist in an optic ganglion of the fly. However, an equivalent map for processing spatial positions of single objects has not been hitherto identified in any arthropod. Crabs can escape directly away from a visual threat wherever the stimulus is located in the 360° field of view. When tested in a walking simulator, the crab Neohelice granulata immediately adjusts its running direction after changes in the position of the visual danger stimulus smaller than 1°. Combining mass and single-cell staining with in vivo intracellular recording, we show that a particular class of motion-sensitive neurons of the crab's lobula that project to the midbrain, the monostratified lobula giants type 1 (MLG1), form a system of 16 retinotopically organized elements that map the 360° azimuthal space. The preference of these neurons for horizontally moving objects conforms the visual ecology of the crab's mudflat world. With a mean receptive field of 118°, MLG1s have a large superposition among neighboring elements. Our results suggest that the MLG1 system conveys information on object position as a population vector. Such computational code can enable the accurate directional control observed in the visually guided behaviors of crabs.