Novel marine metalloprotease-new approaches for inhibition of biofilm formation of Stenotrophomonas maltophilia.

Many marine organisms produce bioactive molecules with unique characteristics to survive in their ecological niches. These enzymes can be applied in biotechnological processes and in the medical sector to replace aggressive chemicals that are harmful to the environment. Especially in the human health sector, there is a need for new approaches to fight against pathogens like Stenotrophomonas maltophilia which forms thick biofilms on artificial joints or catheters and causes serious diseases. Our approach was to use enrichment cultures of five marine resources that underwent sequence-based screenings in combination with deep omics analyses in order to identify enzymes with antibiofilm characteristics. Especially the supernatant of the enrichment culture of a stony coral caused a 40% reduction of S. maltophilia biofilm formation. In the presence of the supernatant, our transcriptome dataset showed a clear stress response (upregulation of transcripts for metal resistance, antitoxins, transporter, and iron acquisition) to the treatment. Further investigation of the enrichment culture metagenome and proteome indicated a series of potential antimicrobial enzymes. We found an impressive group of metalloproteases in the proteome of the supernatant that is responsible for the detected anti-biofilm effect against S. maltophilia. KEY POINTS: • Omics-based discovery of novel marine-derived antimicrobials for human health management by inhibition of S. maltophilia • Up to 40% reduction of S. maltophilia biofilm formation by the use of marine-derived samples • Metalloprotease candidates prevent biofilm formation of S. maltophilia K279a by up to 20.

Morphology and frictional properties of scales of Pseudopus apodus (Anguidae, Reptilia).

In the lizard family Anguidae different levels of limb reduction exist up to a completely limbless body. The locomotion patterns of limbless anguid lizards are similar to the undulating and concertina movements of snakes. Additionally, anguid lizards frequently use a third mode of locomotion, called slide-pushing. During slide-pushing the undulating moving body slides on the ground, while the posterior part of the body is pressed against the substrate. Whereas the macroscopic and microscopic adaptations of snake scales to limbless locomotion are well described, the micromorphology of anguid lizard scales has never been examined. Therefore we studied the macro- and micromorphology of the scales of Pseudopus apodus, an anguid lizard with a snakelike body. In addition, we measured the frictional properties of Pseudopus scales. Our data show that the microstructures of the ventral scales of this anguid lizard are less developed than in snakes. We found, however, a rostro-caudal gradient in macroscopic structuring. Whereas the ventral side of the anterior body was nearly unstructured, the tail had macroscopic longitudinal ridges. Our frictional measurements on rough substrates revealed that the ridges provide a frictional anisotropy: friction was higher in the lateral than in the rostral direction. The observed frictional properties are advantageous for a tail-based slide-pushing locomotion, for which a tail with a high lateral friction is most effective in generating propulsion.

Organotopic organization of the primary Infrared Sensitive Nucleus (LTTD) in the western diamondback rattlesnake (Crotalus atrox).

Pit vipers (Crotalinae) have a specific sensory system that detects infrared radiation with bilateral pit organs in the upper jaw. Each pit organ consists of a thin membrane, innervated by three trigeminal nerve branches that project to a specific nucleus in the dorsal hindbrain. The known topographic organization of infrared signals in the optic tectum prompted us to test the implementation of spatiotopically aligned sensory maps through hierarchical neuronal levels from the peripheral epithelium to the first central site in the hindbrain, the nucleus of the lateral descending trigeminal tract (LTTD). The spatial organization of the anatomical connections was revealed in a novel in vitro whole-brain preparation of the western diamondback rattlesnake (Crotalus atrox) that allowed specific application of multiple neuronal tracers to identified pit-organ-supplying trigeminal nerve branches. After adequate survival times, the entire peripheral and central projections of fibers within the pit membrane and the LTTD became visible. This approach revealed a morphological partition of the pit membrane into three well-defined sensory areas with largely separated innervations by the three main branches. The peripheral segregation of infrared afferents in the sensory epithelium was matched by a differential termination of the afferents within different areas of the LTTD, with little overlap. This result demonstrates a topographic organizational principle of the snake infrared system that is implemented by maintaining spatially aligned representations of environmental infrared cues on the sensory epithelium through specific neuronal projections at the level of the first central processing stage, comparable to the visual system.

Subdigital setae of chameleon feet: friction-enhancing microstructures for a wide range of substrate roughness.

Hairy adhesive systems of microscopic setae with triangular flattened tips have evolved convergently in spiders, insects and arboreal lizards. The ventral sides of the feet and tails in chameleons are also covered with setae. However, chameleon setae feature strongly elongated narrow spatulae or fibrous tips. The friction enhancing function of these microstructures has so far only been demonstrated in contact with glass spheres. In the present study, the frictional properties of subdigital setae of Chamaeleo calyptratus were measured under normal forces in the physical range on plane substrates having different roughness. We showed that chameleon setae maximize friction on a wide range of substrate roughness. The highest friction was measured on asperities of 1 µm. However, our observations of the climbing ability of Ch. calyptratus on rods of different diameters revealed that also claws and grasping feet are additionally responsible for the force generation on various substrates during locomotion.

Non-contaminating camouflage: multifunctional skin microornamentation in the West African Gaboon viper (Bitis rhinoceros).

The West African Gaboon viper (Bitis rhinoceros) has an extraordinary coloration of pale brown and velvety black markings. The velvety black appearance is caused by a unique hierarchical surface structures which was not found on the pale brown scales. In the present study we examined the wettability of the vipers scales by measuring contact angles of water droplets. Velvet black scale surfaces had high static contact angles beyond 160° and low roll-off angles below 20° indicating an outstanding superhydrophobicity. Our calculations showed that the Cassie-Baxter model describes well wettability effects for these surfaces. Self-cleaning capabilities were determined by contaminating the scales with particles and fogging them until droplets formed. Black scales were clean after fogging, while pale scales stayed contaminated. Black scales feature multifunctional structures providing not only water-repellent but also self-cleaning properties. The pattern of nanoridges can be used as a model for surface-active technical surfaces.

3D flow in the venom channel of a spitting cobra: do the ridges in the fangs act as fluid guide vanes?

The spitting cobra Naja pallida can eject its venom towards an offender from a distance of up to two meters. The aim of this study was to understand the mechanisms responsible for the relatively large distance covered by the venom jet although the venom channel is only of micro-scale. Therefore, we analysed factors that influence secondary flow and pressure drop in the venom channel, which include the physical-chemical properties of venom liquid and the morphology of the venom channel. The cobra venom showed shear-reducing properties and the venom channel had paired ridges that span from the last third of the channel to its distal end, terminating laterally and in close proximity to the discharge orifice. To analyze the functional significance of these ridges we generated a numerical and an experimental model of the venom channel. Computational fluid dynamics (CFD) and Particle-Image Velocimetry (PIV) revealed that the paired interior ridges shape the flow structure upstream of the sharp 90° bend at the distal end. The occurrence of secondary flow structures resembling Dean-type vortical structures in the venom channel can be observed, which induce additional pressure loss. Comparing a venom channel featuring ridges with an identical channel featuring no ridges, one can observe a reduction of pressure loss of about 30%. Therefore it is concluded that the function of the ridges is similar to guide vanes used by engineers to reduce pressure loss in curved flow channels.

Snake velvet black: hierarchical micro- and nanostructure enhances dark colouration in Bitis rhinoceros.

The West African Gaboon viper (Bitis rhinoceros) is a master of camouflage due to its colouration pattern. Its skin is geometrically patterned and features black spots that purport an exceptional spatial depth due to their velvety surface texture. Our study shades light on micromorphology, optical characteristics and principles behind such a velvet black appearance. We revealed a unique hierarchical pattern of leaf-like microstructures striated with nanoridges on the snake scales that coincides with the distribution of black colouration. Velvet black sites demonstrate four times lower reflectance and higher absorbance than other scales in the UV-near IR spectral range. The combination of surface structures impeding reflectance and absorbing dark pigments, deposited in the skin material, provides reflecting less than 11% of the light reflected by a polytetrafluoroethylene diffuse reflectance standard in any direction. A view-angle independent black structural colour in snakes is reported here for the first time.

Subdigital and subcaudal microornamentation in Chamaeleonidae--a comparative study.

Locomotion on horizontal and vertical substrates requires effective attachment systems. In three clades of arboreal and rupicolous Iguanidae, Gekkota and Scincidae adhesive systems consisting of microscopic hair-like structures (setae) have been evolved independently. Also the substrate contacting sites on toes and tails of chameleons (Chamaeleonidae) are covered with setae. In the present comparative scanning electron microscopy study, we show that representatives from the chamaeleonid genera Calumma, Chamaeleo, Furcifer, and Trioceros feature highly developed setae that are species-specific and similar on their feet and tail. These 10 µm long, unbranched setae rather resemble those in anoline and scincid lizards than the larger and branched setae of certain gecko species. In contrast to the thin triangular tips of other lizards, all examined species of the genera Furcifer and Calumma and one of the five examined species of the genus Trioceros have spatulate tips. All other examined species of genera Trioceros and Chamaeleo bear setae with narrowed, fibrous tips. Unlike the setae of other lizards, chamaeleonid setal tips do not show any orientation along the axis of the toes, but they are flexible to bend in any direction. With these differences, the chameleon's unique microstructures on the zygodactylous feet and prehensile tail rather increase friction for arboreal locomotion than being a shear-induced adhesive system as setal pads of other lizards.

Potential targets aimed at by spitting cobras when deterring predators from attacking.

When threatened, spitting cobras eject venom towards the face of an aggressor. To uncover the relevant cues used by cobras for face recognition we determined how often artificial targets equipped with or without eyes elicited spitting behavior. In addition, we measured whether and how target shape and size influenced the spitting behavior of cobras. Results show that oval- and round-shaped targets were most effective, while triangles with the same surface area as oval 'face like' targets hardly elicited spitting. The likelihood of spitting depended on neither the presence, the spatial arrangement (horizontal or vertical) nor the surface texture (shiny or matt) of glass eyes. Most likely, cobras do not specifically aim at the eyes of an offender but at the center of the body part closest to them. As this is usually the face of an animal, this strategy will result in at least one eye of the offender being hit most of the time.

Directional sensitivity in the thermal response of the facial pit in western diamondback rattlesnakes (Crotalus atrox).

Recent work published in the accompanying paper used a combination of 3D morphological reconstruction to define optical spread functions and heat transfer physics to study how external heat energy would reach the sensory membrane within the facial pit of pitvipers. The results from all of the species examined indicated asymmetric directional sensitivity, e.g. the pit would preferentially respond to stimuli located below and behind the snake. The present study was intended as a test of these findings through a quantitative neurophysiological analysis of directional sensitivity in the facial pit of the western diamondback rattlesnake, Crotalus atrox. An infrared emitter was positioned through a coordinate system (with varying angular orientations and distances) and the response it evoked measured through neurophysiological recordings of a trigeminal nerve branch composed of the afferents from the sensory membrane of the facial pit. Significant differences were found in the strength of the membrane's neural response to a constant stimulus presented at different orientations (relative to the facial pit opening) and over different distances. The peak sensitivity (at 12 deg above and 20 deg in front of the facial pit opening) was in good agreement with the predicted directional sensitivities based on optical spread functions and 3D topography. These findings support the hypothesis that the topography, and functional performance, of the facial pit has undergone an adaptive radiation within the pit vipers, and that differences in the behavioral ecology of the pit vipers (i.e. terrestrial versus arboreal) are reflected within the facial pits.

Photoreceptor types, visual pigments, and topographic specializations in the retinas of hydrophiid sea snakes.

Sea snakes have evolved numerous anatomical, physiological, and behavioral adaptations to suit their wholly aquatic lifestyle. However, although sea snakes use vision for foraging and mate selection, little is known about their visual abilities. We used microspectrophotometry, light microscopy, and scanning electron microscopy to characterize the retinal photoreceptors of spine-bellied (Lapemis curtus) and horned (Acalyptophis peronii) sea snakes. Both species have three types of visual pigment sensitive to short (SWS; wavelength of maximum absorbance, λmax 428-430 nm), medium (MWS; λmax 496 nm), and long wavelengths of light (LWS; λmax 555-559 nm) in each of three different subtypes of cone-like single photoreceptor. They also possess a cone-like double photoreceptor subtype, both the principal and accessory member of which contain the LWS visual pigment. Conventional rods were not observed, although the MWS photoreceptor may be a "transmuted" rod. We also used stereology to measure the total number and topographic distribution of neurons in the ganglion cell layer of L. curtus, the olive sea snake (Aipysurus laevis), and the olive-headed sea snake (Disteira major). All species have a horizontal visual streak with specialized areas in the nasal and temporal retina. Both L. curtus and D. major also have a specialized area in the ventral retina, which may reflect differences in habitat usage and/or foraging behavior compared to A. laevis. Maximal spatial resolution was estimated at 1.1, 1.6, and 2.3 cycles deg⁻¹ in D. major, L. curtus, and A. laevis, respectively; the superior value for A. laevis may reflect its specialized crevice-foraging hunting technique.

Connections of the medial telencephalic wall in the spotted African Lungfish.

The extent and boundaries of the roof, or pallium, of the telencephalon in lungfishes have been debated for over 30 years, and two hypotheses exist. Proponents of a restricted pallium claim that the medial border of the pallium occurs in a dorsal position and that the entire medial hemispheric wall is formed by the septal nuclei. Proponents of an extended pallium claim that the medial border of the pallium occurs in a more ventral position and that the medial hemispheric wall is divided into a dorsal medial pallium and ventral septal nuclei, as in amphibians. Immunohistochemical data have generally been interpreted to support the hypothesis of an extended pallium, but disagreement still exists. To clarify the extent of the pallium in lungfishes, the connections of the dorsal and ventral divisions of the medial hemispheric wall in the Spotted African Lungfish were examined using a number of neuronal tracers. In amphibians and other tetrapods, the afferent projections to the medial pallium and the septal nuclei differ extensively, as do the commissural routes taken by decussating interhemispheric connections. Although the descending projections of the medial pallium and septal nuclei are very similar to one another in amphibians and other tetrapods, they do differ in that the septal nuclei and the ventral thalamus are extensively interlinked, whereas the medial pallium lacks such connections. These differences also characterize the connections of the dorsal and ventral divisions of the medial hemispheric wall in the Spotted African Lungfish, which supports the hypothesis of an extended pallium. The telencephalic organization in lungfishes thus appears remarkably similar to that in amphibians and reflects a pattern that almost certainly existed in the last common ancestor of lungfishes and tetrapods.

Target tracking during venom 'spitting' by cobras.

Spitting cobras, which defend themselves by streaming venom towards the face and/or eyes of a predator, must be highly accurate because the venom they spit is only an effective deterrent if it lands on the predator's cornea. Several factors make this level of accuracy difficult to achieve; the target is moving, is frequently >1 m away from the snake and the venom stream is released in approximately 50 ms. In the present study we show that spitting cobras can accurately track the movements of a potentially threatening vertebrate, and by anticipating its subsequent (short-term) movements direct their venom to maximize the likelihood of striking the target's eye. Unlike other animals that project material, in spitting cobras the discharge orifice (the fang) is relatively fixed so directing the venom stream requires rapid movements of the entire head. The cobra's ability to track and anticipate the target's movement, and to perform rapid cephalic oscillations that coordinate with the target's movements suggest a level of neural processing that has not been attributed to snakes, or other reptiles, previously.

Spitting cobras adjust their venom distribution to target distance.

If threatened by a human, spitting cobras defend themselves by ejecting their venom toward the face of the antagonist. Circulating head movements of the cobra ensure that the venom is distributed over the face. To assure an optimal distribution of the venom, the amplitudes of head movements should decrease with increasing target distance. To find out whether cobras (Naja pallida and N. nigricollis) adjust their spitting behavior according to target distance we induced spitting from different distances and analyzed their spitting patterns. Our results show that the spray pattern of spitting cobras is not fixed. Instead the snake matches its venom distribution to the size of the target independent of target distance.

Functional bases of the spatial dispersal of venom during cobra "spitting".

Spitting cobras expulse venom toward the face and/or eyes of potential predators as part of their defensive repertoire. Evaluating the accuracy of the cobras is difficult because the spit venom does not land as a point but rather is distributed, in some cases widely, in complex geometric patterns on the surface of the target. The purpose of this study was to explore the functional bases of the venom's spatial distribution. Using a combination of spatial analysis of "caught" venom, morphology, high-speed digital videography, and electromyography (EMG), three hypothesis were evaluated. Two of these hypotheses--that the spatial distribution was due to differential venom pressure produced by the contractile activity of the adductor mandibulae externus superficiali and that the spatial distribution was produced by the morphology of the venom canal within the fang-were both rejected. The third hypothesis--that the spatial distribution was due to rapid rotational movements of the head about the vertebral column--was supported by analyses of EMG activity within the cervical axial muscles and by predictions of venom-distribution patterns based on these cephalic displacements. These results suggest that the ability to "spit" venom is a unique suite of specializations involving both the axial and the cephalic systems.

Sea snakes (Lapemis curtus) are sensitive to low-amplitude water motions.

The sea snake Lapemis curtus is a piscivorous predator that hunts at dusk. Like land snakes, sea snakes have scale sensillae that may be mechanoreceptive, i.e. that may be useful for the detection of water motions produced by prey fish. In addition, inner ear hair cells of sea snakes may also be involved in the detection of hydrodynamic stimuli. We generated water motions and pressure fluctuations with a vibrating sphere. In the test range 50-200 Hz evoked potentials were recorded from the midbrain of L. curtus in response to vibrating sphere stimuli. In terms of water displacement the lowest threshold amplitudes were in the frequency range 100-150 Hz. In this range peak-to-peak water displacement amplitudes of 1.8 microm (at 100 Hz) and 2.0 microm (150 Hz) generated a neural response in the most sensitive animal. Although this low sensitivity may be sufficient for the detection of fish-generated water motions, it makes it unlikely that L. curtus has a special hydrodynamic sense.

Morphology and projection pattern of medial and dorsal pallial neurons in the frog Discoglossus pictus and the salamander Plethodon jordani.

In the frog Discoglossus pictus and the salamander Plethodon jordani, the morphology and axonal projection pattern of neurons in the medial and dorsal pallium were determined by intracellular biocytin labeling. A total of 77 pallial neurons were labeled in the frog and 58 pallial neurons in the salamander. Within the medial pallium (MP) of the frog, four types of neurons were identified on the basis of differences in their axonal projection pattern. Type I neurons have bilateral projections into telencephalic and diencephalic areas; type II neurons have bilateral projections to telencephalic areas and ipsilaterally descending projections to diencephalic regions; type III neurons have only intratelencephalic connections, and a single type IV neuron has ipsilaterally descending projections. The somata of the four types occupy four nonoverlapping zones. Neurons of the dorsal pallium (DP) project exclusively to the ipsilateral MP and to the dorsal edge of the lateral pallium. In the ventral MP of the salamander, neurons have mostly intratelencephalic projections. Neurons in the dorsal MP project bilaterally to diencephalic and telencephalic regions. Neurons in the medial DP project ipsilaterally to the MP, lateral septum, nucleus accumbens, medial amygdala, and the internal granule layer of the olfactory bulb. In five cases, fibers were found in the commissura hippocampi, but in only two cases could these fibers be followed toward the contralateral MP and septum. Neurons in the lateral DP had no contralateral projections; they projected to the ipsilateral MP and in eight cases to the ipsilateral septum as well. Based on similarities of cytoarchitecture and projection pattern in neurons of the MP and DP, it is proposed that both frogs and salamanders have an MP subdivided into a ventral and dorsal portion, and a DP subdivided into a medial and a lateral portion.