In Vivo Imaging and Characterization of Actin Microridges

Actin microridges form labyrinth like patterns on superficial epithelial cells across animal species. This highly organized assembly has been implicated in mucus retention and in the mechanical structure of mucosal surfaces, however the mechanisms that regulate actin microridges remain largely unknown. Here we characterize the composition and dynamics of actin microridges on the surface of zebrafish larvae using live imaging. Microridges contain phospho-tyrosine, cortactin and VASP, but not focal adhesion kinase. Time-lapse imaging reveals dynamic changes in the length and branching of microridges in intact animals. Transient perturbation of the microridge pattern occurs before cell division with rapid re-assembly during and after cytokinesis. Microridge assembly is maintained with constitutive activation of Rho or inhibition of myosin II activity. However, expression of dominant negative RhoA or Rac alters microridge organization, with an increase in distance between microridges. Latrunculin A treatment and photoconversion experiments suggest that the F-actin filaments are actively treadmilling in microridges. Accordingly, inhibition of Arp2/3 or PI3K signaling impairs microridge structure and length. Taken together, actin microridges in zebrafish represent a tractable in vivo model to probe pattern formation and dissect Arp2/3-mediated actin dynamics in vivo.     

Morphology and Phylogeny of Thelohanellus marginatus n. sp. (Myxozoa: Myxosporea), a Parasite Infecting the Gills of the Fish Hypophthalmus marginatus (Teleostei: Pimelodidae) in the Amazon River

Thelohanellus marginatus n. sp., a new myxosporean parasite infecting the primary gill filaments of the teleost fish Hypophthalmus marginatus (Pimelodidae) in the Amazon River, is described on the basis of microscopic and molecular procedures. The parasite forms whitish and ellipsoidal cysts up to 250 μm in diam. Myxospores ellipsoidal with a slightly more pointed anterior end, measuring 17.1 ± 0.6 μm in length, 6.9 ± 0.4 μm in width, and 5.1 ± 0.5 μm in thickness. A single pyriform polar capsule, 9.0 ± 0.3 μm long and 6.1 ± 0.4 μm wide, positioned slightly right to the medial plane in valvular view, contains a polar filament arranged in 4–5 coils. Molecular analysis of the SSU rRNA gene by Maximum Parsimony, Neighbor‐Joining, and Maximum Likelihood revealed the parasite clustering among other myxobolids, namely Henneguya and Myxobolus. Host affinity is supported as an important evolutionary signal for the phylogeny of myxobolids. The parasite here described represents the first record of the genus Thelohanellus Kudo, 1933 from the South American fauna.     

Ortholinea nupchi n. sp. (Myxosporea: Ortholineidae) from the urinary bladder of the cultured olive flounder Paralichthys olivaceus,South Korea

A new myxosporean parasite, Ortholinea nupchi n. sp. (Myxozoa; Bivalvulida), was isolated from the urinary bladder of the olive flounder Paralichthys olivaceus cultured on Jeju Island, Korea. Mature spores were subspherical in the valvular and apical views and ellipsoidal in the sutural view. The spores measured 7.6 ± 0.5 μm in length, 6.7 ± 0.3 μm in thickness, and 7.3 ± 0.5 μm in width. Two pyriform polar capsules measured 3.2 ± 0.1 μm in length and 2.7 ± 0.1 μm in width and were located at the same level at the anterior half of the myxospores. The suture line was straight in the middle of the spores, and the surface ridges ranged between five and seven, forming an intricate pattern. The result of the 18S rDNA comparison showed ≤ 93.0% similarity with other Ortholinea species. The phylogenetic tree demonstrated that O. nupchi n. sp. was closest to O. auratae and clustered with oligochaete-infecting myxosporeans (OIM) having urinary system infection tropism. Based on the comparison of environmental and host factors in the phylogenetic groups of the OIM clade, we propose that the infection of O. nupchi n. sp. originated from marine oligochaetes.     

Comparison of microridges in juvenile and adult sunfish,Lepomis gibbosus

Microridges are F-actin-based surface protrusions of the superficial layer cells of fish epidermis. Microridge patterns progress in complexity during fish embryogenesis, often transitioning from abundant surface microvilli to the classical fingerprint arrangement. This progression suggests pattern changes may also occur during later stages of fish development. Fluorescent labelling of F-actin and morphometric analysis were therefore used to assess changes in epidermal microridge patterns in juvenile and adult sunfish (Lepomis gibbosus). The microridge patterns found in adult pumpkinseed were similar to that described for many fishes, consisting of whorls or complex multi-branched ridges. The microridge patterns of the scales from three different-sized groups of juvenile pumpkinseed were distinctly different from that of adult, however, and were present mainly as unbranched concentric or nearly concentric rings in the two larger juvenile groups. In the smallest juveniles, microridges were often apparent as fragmented ridges with abundant actin puncta. Larger juveniles sometimes displayed mixed patterns, with some microridges similar to that of both adult and juvenile patterns. The results show a transition from simple microridge patterns in juvenile pumpkinseeds to distinctly different, diverse and more complex patterns in adults. The different pattern types may reflect particular microridge functions relevant to fish size and age.     

Textures and traction: how tube‐dwelling polychaetes get a leg up

By controlling the traction between its body and the tube wall, a tube‐dwelling polychaete can move efficiently from one end of its tube to the other, brace its body during normal functions (e.g., ventilation and feeding), and anchor within its tube avoiding removal by predators. To examine the potential physical interaction between worms and the tubes they live in, scanning electron microscopy was used to reveal and quantify the morphology of worm bodies and the tubes they produce for species representing 13 families of tube‐dwelling polychaetes. In the tubes of most species there were macroscopic or nearly macroscopic (~10 μm–1 mm) bumps or ridges that protruded slightly into the lumen of the tube; these could provide purchase as a worm moves or anchors. At this scale (~10 μm‐1 mm), the surfaces of the chaetal heads that interact with the tube wall were typically small enough to fit within spaces between these bumps (created by the inward projection of exogenous materials incorporated into the tube wall) or ridges (made by secretions on the interior surface of the tube). At a finer scale (0.01–10 μm), there was a second overlap in size, usually between the dentition on the surfaces of chaetae that interact with the tube walls and the texture provided by the secreted strands or microscopic inclusions of the inner linings. These linings had a surprising diversity of micro‐textures. The most common micro‐texture was a “fabric” of secreted threads, but there were also orderly micro‐ridges, wrinkles, and rugose surfaces provided by microorganisms incorporated into the inner tube lining. Understanding the fine structures of tubes in conjunction with the morphologies of the worms that build them gives insight into how tubes are constructed and how worms live within them.     

Cargo properties play a critical role in myosin Va-driven cargo transport along actin filaments

High-resolution experiments revealed that a single myosin-Va motor can transport micron-sized cargo on actin filaments in a stepwise manner. However, intracellular cargo transport is mediated through the dense actin meshwork by a team of myosin Va motors. The mechanism of how motors interact mechanically to bring about efficient cargo transport is still poorly understood. This study describes a stochastic model where a quantitative understanding of the collective behaviors of myosin Va motors is developed based on cargo stiffness. To understand how cargo properties affect the overall cargo transport, we have designed a model in which two myosin Va motors were coupled by wormlike chain tethers with persistence length ranging from 10 to 80 nm and contour length from 100 to 200 nm, and predicted distributions of velocity, run length, and tether force. Our analysis showed that these parameters are sensitive to both the contour and persistence length of cargo. While the velocity of two couple motors is decreased compared to a single motor (from 531 ± 251 nm/s to as low as 318 ± 287 nm/s), the run length (716 ± 563 nm for a single motor) decreased for short, rigid tethers (to as low as 377 ± 187 μm) and increased for long, flexible tethers (to as high as 1.74 ± 1.50 μm). The sensitivity of processive properties to tether rigidity (persistence length) was greatest for short tethers, which caused the motors to exhibit close, yet anti-cooperative coordination. Motors coupled by longer tethers stepped more independently regardless of tether rigidity. Therefore, the properties of the cargo or linkage must play an essential role in motor-motor communication and cargo transport.     

A New Freshwater Amoeba: Cochliopodium pentatrifurcatum n. sp. (Amoebozoa,Amorphea)

Cochliopodium pentatrifurcatum n. sp. (ATCC^© 30935^TM) is described based on light microscopic morphology, fine structure, and molecular genetic evidence. Cochliopodium pentatrifurcatum n. sp. (length ~ 25 μm) is characterized by surface microscales (0.3 μm tall) containing a circular porous base (~ 0.6 μm diam.) with a thin peripheral rim. Five radially arranged feet, emanating from the base, support a short central column terminating apically as a funnel‐shaped collar (~ 0.5 μm diam.) composed of five radial, trifurcate rays extending from the center toward a thin peripheral rim. The central spine is 0.5–0.6 μm long. The comparative morphologies and combined molecular genetic evidence, SSU‐rDNA and COI, indicate that the new species falls in a clade sufficiently different from other species to suggest that it is a valid new species.     

Habitat Associations and Community Structure of Dipterocarps in Response to Environment and Soil Conditions in Brunei Darussalam,Northwest Borneo

Plant habitat associations are well documented in Bornean lowland tropical forests, but few studies contrast the prevalence of associations across sites. We examined habitat associations and community composition of Dipterocarpaceae trees in two contrasting Bornean lowland mixed dipterocarp forests separated by approximately 100 km: Andulau (uniform topography, lower altitudinal range, sandy soils) and Belalong (highly dissected topography, higher altitudinal range, clay‐rich soils). Dipterocarpaceae trees ≥ 1 cm diameter at breast height (dbh) were censused in 20‐m wide belt transects established along topographic gradients at each site. Dipterocarp density, evenness, species richness, and diversity were significantly higher at Andulau than Belalong. Significant site associations (with either Andulau or Belalong) were detected for 19 (52%) of the 37 dipterocarp species tested. Dipterocarpaceae community composition at Belalong correlated with soil nutrient concentrations as well as measures of vegetation and topographic structure, but community composition at Andulau correlated with fewer habitat variables. Within each site, dipterocarp density, species richness, and diversity were consistently higher on ridges than in slopes and valleys. Significant within‐site associations to topographic habitats were less common at Andulau (10% of species tested) than at Belalong (15%). We conclude that edaphic and other environmental factors influence dipterocarp community composition at a local scale, and are more important drivers of community structure in the more variable environment at Belalong. Species richness and diversity of dipterocarps on small plots, however, were higher at Andulau, suggesting that factors other than environmental heterogeneity contribute to contrasts in dipterocarp tree species richness at small scales.     

Molecular and Morphometric Characteristics of Ceratomyxa hamour n. sp. (Myxosporea: Bivalvulida) Infecting the Gallbladder of the Orange‐spotted Grouper Epinephelus coioides from the Arabian Gulf,Saudi Arabia

Ceratomyxa hamour n. sp. was found to infect the gallbladder of the orange‐spotted grouper, Epinephelus coioides located off the Saudi Arabian coast of the Arabian Gulf. The infection was reported as a free‐floating spore in the bile, and pseudoplasmodia were not observed. Mature spores were crescent‐shaped and measured on average 7 μm in length and 16 μm in thickness. The polar capsule, meanwhile, had length to width measurements of 4 μm and 3 μm on average. A periodical survey was conducted throughout a sampling period between December 2012 and December 2013, with the results showing that the parasite was present throughout the year with a mean prevalence of 32.6%. The objective of this study was to characterize this new species based on its morphological and molecular differences from previously described species. Molecular analysis based on the partial sequence of the SSU rDNA gene, showed the highest similarity (97.8%) to Ceratomyxa buri, reported in the cultured yellow tail Seriola quinqueradiata in Japan. Indeed, C. buri and the new species described here formed an individual cluster with a high degree of bootstrap support. This is the first reported species of genus Ceratomyxa from the Arabian Gulf fishes off Saudi Arabia.     

The spermatozoon of Mengenilla moldrzyki (Strepsiptera,Mengenillidae): Ultrastructure and phylogenetic considerations

Even though the spermatozoa of several strepsipteran species were described earlier, no data were available for the basal family Mengenillidae. Well-fixed material of the recently described Tunisian species Mengenilla moldrzyki was used for a detailed examination of the sperm ultrastructure. The total length is c. 30 μm. The head region contains a conical acrosome vesicle (0.3-0.35 μm) and an elongated nucleus (7.3 μm) with dense chromatin. Some granular material along with a uniformely dense centriole adjunct and two mitochondrial derivatives are visible at the posterior end of the nucleus. The material of the centriole adjunct does not extend along the flagellum and accessory bodies are absent. The mitochondrial derivatives are elongated structures crossed by a longitudinal crista but lacking parallel transverse cristae and paracrystalline material in the dense matrix. The mitochondrial derivatives gradually reduce their size and end at the most posterior tail region. The flagellar axoneme has a 9 + 9 + 2 pattern and originates beneath the nucleus. In the terminal tail region the axoneme gradually disintegrates. Despite the extreme specialization of the endoparasitc group, strepsipteran spermatozoa are mostly characterized by plesiomorphies. The pattern within the order is largely uniform, but Mengenilla displays several apomorphic features compared to the presumptive strepsipteran groundplan (e.g., absence of crystallizations and cristae in the mitochondrial derivatives). The subdivision of the intertubular material into two compartments with a dense beak-like structure adhering to the tubular wall supports a clade Coleopterida (=Strepsiptera + Coleoptera) + Neuropterida.     

Morphological and molecular analysis of Henneguya lagunensis n. sp. (Cnidaria,Myxosporea) parasitizing the gills of Eugerres brasilianus from Brazil

Plasmodia containing myxospores belonging to the genus Henneguya Thélohan, 1892 were found in the gills of Eugerres brasilianus (Cuvier, 1830). Despite the economic importance, few parasitological studies have been done with this species. We describe Henneguya lagunensis n. sp. using morphological and molecular data. The mature myxospores were rounded, measuring 29.1 ± 2.2 μm in total length, 8.2 ± 1.0 μm in body length, 7.9 ± 0.2 μm in body width, 20.7 ± 2.4 μm in tail length and 4.8 ± 1.0 μm in thickness. The polar capsules measured 3.3 ± 0.4 in length and 1.7 ± 0.3 μm in width. Polar filaments had 4–5 turns, helical. Phylogenetic analysis showed Henneguya lagunensis n. sp. as a sister species of Henneguya cynoscioni Dyková, Buron, Roumillat and Fiala, 2011, within a clade that contained mostly Henneguya species that parasitize marine fish of the order Perciformes. This is the first report of a species of Henneguya parasitizing Eugerres brasilianus.     

Actin filaments in microridges of the oral mucosal epithelium in the carp Cyprinus carpio

Summary Actin filaments in the microridges on the surface of the fish oral mucosa taken from Cyprinus carpio were examined by electron microscopy after detergent extraction and decoration with myosin subfragment 1. After extraction with saponin, an irregular and densely packed meshwork of actin filaments was observed in the bases of the microridges, just lateral to the tight junctions with their fibrous undercoats. Actin filaments formed cores in the microridges and numerous linkages were seen between the filaments and the plasma membrane. Extraction with Triton X-100 and decoration with myosin subfragment 1 showed the ends of the actin filaments to be associated with the plasma membrane of the microridges, and in the bases of microridges the filament ends were anchored to intermediate filaments. Some actin filaments interconnected with the fibrous undercoats of the tight junctions. On the basis of these observations, the mechanism of the formation of microridges, including their pattern, is discussed.     

The Kinetics of Mechanically Coupled Myosins Exhibit Group Size-Dependent Regimes

Naturally occurring groups of muscle myosin behave differently from individual myosins or small groups commonly assayed in vitro. Here, we investigate the emergence of myosin group behavior with increasing myosin group size. Assuming the number of myosin binding sites (N) is proportional to actin length (L) (N = L/35.5 nm), we resolve in vitro motility of actin propelled by skeletal muscle myosin for L = 0.2–3 μm. Three distinct regimes were found: L < 0.3 μm, sliding arrest; 0.3 μm ≤ L ≤ 1 μm, alternation between arrest and continuous sliding; L > 1 μm, continuous sliding. We theoretically investigated the myosin group kinetics with mechanical coupling via actin. We find rapid actin sliding steps driven by power-stroke cascades supported by postpower-stroke myosins, and phases without actin sliding caused by prepower-stroke myosin buildup. The three regimes are explained: N = 8, rare cascades; N = 15, cascade bursts; N = 35, continuous cascading. Two saddle-node bifurcations occur for increasing N (mono → bi → mono-stability), with steady states corresponding to arrest and continuous cascading. The experimentally measured dependence of actin sliding statistics on L and myosin concentration is correctly predicted.     

Mechanism of Calponin Stabilization of Cross-Linked Actin Networks

The actin-binding protein calponin has been previously implicated in actin cytoskeletal regulation and is thought to act as an actin stabilizer, but the mechanism of its function is poorly understood. To investigate this underlying physical mechanism, we studied an in vitro model system of cross-linked actin using bulk rheology. Networks with basic calponin exhibited a delayed onset of strain stiffening (10.0% without calponin, 14.9% with calponin) and were able to withstand a higher maximal strain before failing (35% without calponin, 56% with calponin). Using fluorescence microscopy to study the mechanics of single actin filaments, we found that calponin increased the flexibility of actin filaments, evident as a decrease in persistence length from 17.6 μm without to 7.7 μm with calponin. Our data are consistent with current models of affine strain behavior in semiflexible polymer networks, and suggest that calponin stabilization of actin networks can be explained purely by changes in single-filament mechanics. We propose a model in which calponin stabilizes actin networks against shear through a reduction of persistence length of individual filaments.     

Platelet‐rich plasma inhibits osteoblast apoptosis and actin cytoskeleton disruption induced by gingipains through upregulating integrin β1

The aim of this study was to explore the effects of platelet‐rich plasma on gingipain‐caused changes in cell morphology and apoptosis of osteoblasts. Mouse osteoblasts MC3T3‐E1 cells were treated with gingipain extracts from Porphyromonas gingivalis in the presence or absence of platelet‐rich plasma. Apoptosis was detected with terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling staining. F‐actin was determined by phalloidin‐fluorescent staining and observed under confocal microscopy. Western blot analysis was used to detect integrin β1, F‐actin, and G‐actin protein expressions. A knocking down approach was used to determine the role of integrin β1. The platelet‐rich plasma protected osteoblasts from gingipain‐induced apoptosis in a dose‐dependent manner, accompanied by upregulation of integrin β1. Platelet‐rich plasma reversed the loss of F‐actin integrity and decrease of F‐actin/G‐actin ratio in osteoblasts in the presence of gingipains. By contrast, the effects of platelet‐rich plasma were abrogated by knockdown of integrin β1. The platelet‐rich plasma failed to reduce cell apoptosis and reorganize the cytoskeleton after knockdown of integrin β1. In conclusion, platelet‐rich plasma inhibits gingipain‐induced osteoblast apoptosis and actin cytoskeleton disruption by upregulating integrin β1 expression.     

Ultrastructure of grey mullet (Mugil cephalus,Linnaeus, 1758) spermatozoa as revealed from light,scanning and transmission electron microscopy

Spermatozoa morphology and fine structure were studied in the grey mullet, Mugil cephalus using light, scanning and transmission electron microscopy. Light microscopy observations indicate a semi‐cystic type of spermatogenesis in the testis. The electron microscopy micrograph showed that the spermatozoon of M. cephalus is uniflagellated (total length 5.78 ± 1.26 μm), differentiated into an ovoid‐shaped head without acrosome (1.80 ± 0.35 μm in length and 1.91 ± 0.30 μm in diameter), with a short midpiece and a long cylindrical flagellar tail (length 3.60 ± 0.50 μm). The midpiece is characterized by the presence of four to five vacuoles, a cytoplasmic canal, two centriole and two spherical mitochondria having a flat type of cristae. Chromatin granules of the nucleus form an electron‐dense homogeneous mass. The flagellum consists of nine peripheral microtubules and a central pair (9 + 2) surrounded by the plasma membrane with side fins. The results confirm that spermatozoa of M. cephalus are perciform or teleostean type II. Information generated from the present study will be useful in taxonomic classification, cryopreservation and breeding work.     

Cofilin increases the bending flexibility of actin filaments: implications for severing and cell mechanics

We determined the flexural (bending) rigidities of actin and cofilactin filaments from a cosine correlation function analysis of their thermally driven, two-dimensional fluctuations in shape. The persistence length of actin filaments is 9.8 μm, corresponding to a flexural rigidity of 0.040 pN μm². Cofilin binding lowers the persistence length ∼5-fold to a value of 2.2 μm and the filament flexural rigidity to 0.0091 pN μm². That cofilin-decorated filaments are more flexible than native filaments despite an increased mass indicates that cofilin binding weakens and redistributes stabilizing subunit interactions of filaments. We favor a mechanism in which the increased flexibility of cofilin-decorated filaments results from the linked dissociation of filament-stabilizing ions and reorganization of actin subdomain 2 and as a consequence promotes severing due to a mechanical asymmetry. Knowledge of the effects of cofilin on actin filament bending mechanics, together with our previous analysis of torsional stiffness, provide a quantitative measure of the mechanical changes in actin filaments associated with cofilin binding, and suggest that the overall mechanical and force-producing properties of cells can be modulated by cofilin activity.     

Steinernema brazilense n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from Mato Grosso, Brazil

A new entomopathogenic nematode, Steinernema brazilense n. sp., was isolated from a single soil sample collected from a natural forest in Mato Grosso do Sul state, Brazil. S. brazilense n. sp. is characterized morphologically by features of infective juveniles (IJ), males and females. For the IJ, body length averaging 1157 (1023-1284) μm, distance from anterior end to excretory pore 95 (87-102) μm, from anterior end to end of esophagus 148 (139-153) μm, tail length 85 (80-104) μm, D% and E% values 63 (58-70) and 106 (95-118.0), respectively. Lateral field pattern variable; the formula for the arrangement of ridges from head to tail is: 2, 4, 6, 8, 6, 2. For the male, the diagnostic characters include spicule averaging 83 (75-89) μm; D% about 65; the ratio SW% about 192. The length of spicule head is greater than width. Lateral field with one narrow ridge. First generation females are characterized by the presence of a ventral postanal swelling. S. brazilense n. sp. is morphologically close to Steinernema diaprepesi. It can be differentiated from S.diaprepesi by its longer IJ body length (1157 vs 1002 μm), longer distance from anterior end to excretory pore (110 vs 75 μm), a longer tail length (103 vs 83 μm); males of the new species with longer spicule (83 vs 79 μm). The new species can be distinguished further from other members of Steinernema glaseri group by characteristics of rDNA of ITS and D2D3 regions.     

Fine structure of Chloromyxum menticirrhi n. sp. (Myxozoa) infecting the urinary bladder of the marine teleost Menticirrhus americanus (Sciaenidae) in Southern Brazil

A myxosporidian was found in the urinary bladder of the teleost Menticirrhus americanus Linnaeus, 1758 (Sciaenidae) collected from the South Atlantic coast of Brazil. Polysporic amoeboid plasmodia containing sporoblasts, developing pansporoblasts and spores were free in the bladder lumen. The prevalence of infection was 17.64% (15/85). Unfixed spores were spherical to subspherical, on average 10.5 μm long, 9.8 μm wide and 10.1 μm thick (n=25), and fixed spores measured 10.1×9.5×9.7 μm. The two spore valves were of equal size and each possessed prominent sutural lines and about 41 (37–45) surface ridges aligned parallel with the suture line. These ridges gave transverse sections a cog-wheel-like outline. The spores contained four pyriform polar capsules of equal size (3.20×2.0 μm) (n=25) (fixed), each with a polar filament having 3–4 (rarely 5) coils. The binucleate sporoplasm was irregular in shape, with granular matrix and randomly distributed dense bodies. The shape and dimensions of the spore, as well as the number, position and arrangement of the surface ridges, polar capsules and polar filament indicate that this is a new species, herein designated Chloromyxum menticirrhi. The gill, liver, gall bladder and intestine of the host showed no abnormalities.