Sucker Ring Research Articles

Evaluation of schistosoma mansoni nervous system using confocal laser electron microscopy: nerve sensilla and FMRFamide while referring to F-actin abundance

ABSTRACT The chaetotaxy distribution pattern of surface nerve sensilla on the surface of both Schistosoma mansoni and S. haematobium was studied after exposure to silver nitrate treatment followed by light. This distribution pattern could not be achieved earlier due to namelessness, crowded nerve sensilla and twist of most parasites. In S. mansoni, silver impregnation technique displays nerves as ventral nerve cord, ventral sucker nerve that branches to three rings which innervate the ventral sucker. This technique shows the oral sucker ring, ring commissures and bipolar or multipolar nerve cells detected dorsally. In S. haematbium, nerves stained in S. mansoni were also stained in addition to the oral nerve and oral sucker ring. Furthermore, a number of crucial nerves were identified and their innervations to relevant organs were thoroughly examined. A confocal anti-FMRF amide technique was applied to whole-mount worms of both species. In addition to the general tegumental muscles stated previously and stated in this research specific nerves were also described and referred to a specific organ that assumed to innervate. In conclusion, the findings of the current study differentiate between S. mansoni and S. mansoni using chaetotaxy map as a step for spotlighting on FMRF amidergic components as antihelmintic target in future.

Thermomechanical and Morphological Properties of Loligo vulgaris Squid Sucker Ring Teeth.

Climate change is accelerating the increase of temperatures across the planet and resulting in the warming of oceans. Ocean warming threatens the survival of many aquatic species, including squids, and has introduced physiological, behavioral, and developmental changes, as well as physical changes in their biological materials composition, structure, and properties. Here, we characterize and analyze how the structure, morphology, and mechanical properties of European common squid Loligo vulgaris sucker ring teeth (SRT) are affected by temperature. SRT are predatory teethed structures located inside the suction cups of squids that are used to capture prey and are composed of semicrystalline structural proteins with a high modulus (GPa-range). We observed here that this biological material reversibly softens with temperature, undergoing a glass transition at ∼35°C, to a MPa-range modulus. We analyzed the SRT protein nanostructures as a function of temperature, as well as microscale and macroscale morphological changes, to understand their impact in the material properties. The results suggested that even small deviations from their habitat temperatures can result in significant softening of the material (up to 40% in modulus loss). Temperature changes following recent global climate trends and predictions might affect environmental adaptation in squid species and pose emerging survival challenges to adapt to increasing ocean temperatures.

The shape of sucker ring teeth in the hectocotylized part of kisslip cuttlefish (Sepia lycidas)

The shape of sucker ring teeth in the hectocotylized part of kisslip cuttlefish (Sepia lycidas)

Bioinspired Squid Peptides─A Tale of Curiosity-Driven Research Leading to Unforeseen Biomedical Applications.

The molecular design of many peptide-based materials originates from structural proteins identified in living organisms. Prominent examples that have garnered broad interdisciplinary research interest (chemistry, materials science, bioengineering, etc.) include elastin, silk, or mussel adhesive proteins. The critical first steps in this type of research are to identify a convenient model system of interest followed by sequencing the prevailing proteins from which these biological structures are assembled. In our laboratory, the main model systems for many years have been the hard biotools of cephalopods, particularly their parrot-like tough beak and their sucker ring teeth (SRT) embedded within the sucker cuptions that line the interior surfaces of their arms and tentacles. Unlike the majority of biological hard tissues, these structures are devoid of biominerals and consist of protein/polysaccharide biomolecular composites (the beak) or, in the case of SRT, are entirely made of proteins that are assembled by supramolecular interactions.In this Account, we chronicle our journey into the discovery of these intriguing biological materials. We initially focus on their excellent mechanical robustness followed by the identification and sequencing of the structural proteins from which they are built, using the latest "omics" techniques including next-generation sequencing and high-throughput proteomics. A common feature of these proteins is their modular architecture at the molecular level consisting of short peptide repeats. We describe the molecular design of these peptide building blocks, highlighting the consensus motifs identified to play a key role in biofabrication and in regulating the mechanical properties of the macroscopic biological material. Structure/property relationships unveiled through advanced spectroscopic and scattering techniques, including Raman, infrared, circular dichroism, and NMR spectroscopies as well as wide-angle and small-angle X-ray scattering, are also discussed.We then present recent developments in exploiting the discovered molecular designs to engineer peptides and their conjugates for promising biomedical applications. One example includes short peptide hydrogels that self-assemble entirely under aqueous conditions and simultaneously encapsulate large macromolecules during the gelation process. A second example involves peptide coacervate microdroplets produced by liquid-liquid phase separation. These microdroplets are capable of recruiting and delivering large macromolecular therapeutics (genes, mRNA, proteins, peptides, CRISPR/Cas 9 modalities, etc.) into mammalian cells, which introduces exciting prospects in cancer, gene, and immune therapies.This Account also serves as a testament to how curiosity-driven explorations, which may lack an obvious practical goal initially, can lead to discoveries with unexpected and promising translational potential.

Morphological and Genetic Differentiation of Loliolus (Nipponololigo) beka (Cephalopoda: Loliginidae) in Coastal China

The population genetic structure of 211 samples of Loliolus (Nipponololigo) beka, which were selected from across seven geographic localities—in the Bohai Sea, the Yellow Sea and the East China Sea—were analyzed using mitochondrial COI and 16S rRNA gene markers. Phylogenetic trees and a haplotype network both showed that the L. (N.) beka localities were genetically distinct, forming two homogeneous lineages: Lineage A and Lineage B. The results of an AMOVA showed that the genetic variation in the L. (N.) beka populations was dominated by the genetic variation between the two lineages, and both the genetic distance and genetic differentiation indices indicated that the genetic differentiation between the two lineages of L. (N.) beka in Chinese waters had reached the level of species divergence. To further confirm the differences between the two lineages shown in the molecular results, we performed a detailed analysis based on morphometric observations and a multivariate statistical analysis to compare the morphology characteristics of Lineage A and Lineage B. The results showed that there were significant differences (p < 0.05) in the ventral mantle length (VML); the mantle width index (MWI); the fin width index (FWI); the head length index (HLI); the left Arm IV length index (LALI4), the right Arm III length index (RALI3), the right Arm IV length index (RALI4), and the hectocotylized proportion of the left Arm IV length (HcL%) between the two lineages. The differences between the two lineages were also supported by the analysis results for the number of sucker ring teeth. Accordingly, the results of the morphological analysis further confirmed the molecular analysis and provided additional evidence for the presence of the cryptic species of L. (N.) beka in the coastal areas of China.

Molecular Insights into the Self-Assembly of Block Copolymer Suckerin Polypeptides into Nanoconfined β-Sheets.

Suckerin in squid sucker ring teeth is a block-copolymer peptide comprised of two repeating modules-the alanine and histidine-rich M1 and the glycine-rich M2. Suckerin self-assemblies display excellent thermo-plasticity and pH-responsive properties, along with the high biocompatibility, biodegradability, and sustainability. However, the self-assembly mechanism and the detailed role of each module are still elusive, limiting the capability of applying and manipulating such biomaterials. Here, the self-assembly dynamics of the two modules and two minimalist suckerin-mimetic block-copolymers, M1-M2-M1 and M2-M1-M2, in silico is investigated. The simulation results demonstrate that M2 has a stronger self-association but weaker β-sheet propensities than M1. The high self-assembly propensity of M2 allows the minimalist block-copolymer peptides to coalesce with microphase separation, enabling the formation of nanoconfined β-sheets in the matrix formed by M1-M2 contacts. Since these glycine-rich fragments with scatted hydrophobic and aromatic residues are building blocks of many other block-copolymer peptides, the study suggests that these modules function as the "molecular glue" in addition to the flexible linker or spacer to drive the self-assembly and microphase separation. The uncovered molecular insights may help understand the structure and function of suckerin and also aid in the design of functional block-copolymer peptides for nanotechnology and biomedicine applications.

Species identification of two Loliginidae cryptic species in China Seas with morphological and molecular methods

Uroteuthis chinensis and Uroteuthis edulis are morphologically similar squids and commercially important for Indo-Pacific Ocean fisheries. Hence, species identification is vital for them because of their similarity. In order to distinguish the two species, two methods (teeth shape of arm sucker ring and DNA sequence) were adopted. In the study, 341 individuals (210 U. chinensis and 131 U. edulis) from different locations of China Seas were used. The two identification methods (teeth shape of arm sucker ring and DNA) yielded inconsistent identification results for 14% of specimens. The high matching rate to DNA results indicated that the identification method utilizing the teeth shape of arm sucker ring was an ideal method. The ratio of Mantle Width (MW) to Mantle length (ML) was not suitable for identification because of the overlapping ratio range between the two species. The ratio of MW to ML showed sharp variations among different ML groups, indicating the allometric growth in both species. The Principal Component Analysis (PCA) and Step-wise Discriminant Analysis (SDA) results showed that the morphological similarity between the two species from the same area was greater than that of the species from different areas. SDA results also showed that the validation rates of three groups (U. edulis in East China Sea, U. edulis in South China Sea, and U. chinensis in South China Sea) were above 75%. Phylogenetic analysis based on COI showed that U. edulis in the study formed two clades, whereas U. chinensis just formed one clade. U. chinensis was only found in the south of 25°N. The finding should be confirmed through a large amount of samples in East China Sea. The Haplotype network indicate that U. edulis in ECS and SCS were both independent evolutionary units. These cladogenetic events seem to be related to habitat discontinuities, oceanographic and geographic processes that build the barriers for gene flow of U. edulis.

Evolutionary development of the cephalopod arm armature: a review

The cephalopod arm armature is certainly one of the most important morphological innovations responsible for the evolutionary success of the Cephalopoda. New palaeontological discoveries in the recent past afford to review and reassess origin and homology of suckers, sucker rings, hooks, and cirri. Since a priori character state reconstructions are still ambiguous, we suggest and discuss three different evolutionary scenarios. Each of them is based on the following assumptions: (1) Neocoleoidea uniting extant Decabrachia and Octobrachia is monophyletic (= proostracum-bearing coleoids); (2) extinct Belemnitida and Diplobelida are stem decabrachians; (3) proostracum-less coleoids (Hematitida, Donovaniconida, Aulacoceratida) represent stem-neocoleoids; (4) Ammonoidea and Bactritoidea are stem coleoids. We consider a scenario where belemnoid hooks derived from primitive suckers as well-supported. Regarding belemnoid hooks and suckers as homologues implies that belemnoid, oegopsid, and probably ammonoid arm hooks arose through parallel evolution. Our conclusions challenge the widespread opinion, whereupon belemnoid hooks evolved de novo, and instead support earlier ideas formulated by Sigurd von Boletzky.

Morphology of the suckers for hunting behavior in broadclub cuttlefish (Sepia latimanus)

AbstractCuttlefish has eight arms and two tentacles that it uses to catch prey. Cuttlefish finds prey, extends its tentacles, catches the prey using tentacular clubs, and retracts the prey. After retraction, the cuttlefish holds and transfers the prey to its mouth using its arms. Since this efficient unique predation is one of the significant factors that have made the cuttlefish a keystone species in marine food ecosystems, many behavioral studies have been conducted. However, the morphological benefit of the sucker on the arms and tentacles for capturing behavior is poorly understood. Here, we observed the relationship between the morphology and function of the arm and tentacular suckers of the broadclub cuttlefish qualitatively and quantitatively. The arm sucker protrudes from the arm surface and has a shorter sucker pedicel length than that of the tentacular sucker. The longest point of the inner sucker ring of the arm sucker is on the aboral side. These are adaptive features to hold and transfer prey effectively from the arms to the mouth. In contrast, the tentacular sucker is stored in the groove of the tentacular club and has a longer sucker stalk length than that of the arm sucker. The longest point of the inner sucker ring of the tentacular sucker is on the outer direction of the holdfast of the tentacular stalk. These are adaptive features for the rapid retraction of moving prey. The results indicate that cuttlefish's arm and tentacular sucker structures reflect their practical use for prey capture.

Characterizing and Controlling Nanoscale Self-Assembly of Suckerin-12.

Structural proteins such as "suckerins" present promising avenues for fabricating functional materials. Suckerins are a family of naturally occurring block copolymer-type proteins that comprise the sucker ring teeth of cephalopods and are known to self-assemble into supramolecular networks of nanoconfined β-sheets. Here, we report the characterization and controllable, nanoscale self-assembly of suckerin-12 (S12). We characterize the impacts of salt, pH, and protein concentration on S12 solubility, secondary structure, and self-assembly. In doing so, we identify conditions for fabricating ∼100 nm nanoassemblies (NAs) with narrow size distributions. Finally, by installing a noncanonical amino acid (ncAA) into S12, we demonstrate the assembly of NAs that are covalently conjugated with a hydrophobic fluorophore and the ability to change self-assembly and β-sheet content by PEGylation. This work presents new insights into the biochemistry of suckerin-12 and demonstrates how ncAAs can be used to expedite and fine-tune the design of protein materials.

Using statolith shape analysis to identify five commercial Loliginidae squid species in Chinese waters

Identification of squids in the family Loliginidae is a time-consuming exercise because of the highly overlapping distributions of species and their overall similarity (fin shape and size, sucker ring dentition, and color). Identifying squid based on statolith morphology is considered more accurate than identifying species based on beaks or gladius morphology. We report and compare the statolith shape of five commercially Loliginidae squid (Uroteuthis (Photololigo) duvaucelii, U. edulis, U. chinensis, Loliolus beka, L. japonica) to determine how well these structures discriminate species. Based on statolith morphology, variation in the lateral and dorsal domes enables an 84.8% success rate at classifying species. Environmental factors correlate with statolith shape, and for vertically migrating squid, statolith relative size decreases with increased depth of habitation. Statolith morphology can be used to effectively and accurately identify species of Loliginidae squid occurring in Chinese waters, and may prove valuable for identifying and managing squid resources.

Marine Structural Protein Stability Induced by Hofmeister Salt Annealing and Enzymatic Cross-Linking.

The Humboldt squid is one of the fiercest marine predators thanks in part to its sucker ring teeth that are biopolymer blends of a protein isoform family called suckerin with compression strength that rivals silkworm silk. Here, we focus on the popular suckerin-12 isoform to understand what makes the secondary structure of this biopolymer different in water and the potential role of diverse physical and chemical cross-linkings. By choosing a salt post-treatment, in accordance with the Hofmeister series, we achieved film stability with salt annealing that is comparable to chemical cross-links. By correlating the film morphology with the protein secondary structure changes, suckerin-12 films were shown to contract upon treatment with kosmotropic salts and exhibited increased stability in water. These changes are related to the rearrangement of suckerin-12 secondary structure from random coils and helices to β-sheets. Overall, understanding secondary structure changes caused by aqueous and ionic environments can be instructive for the tuning of the suckerin film sclerotization, its conversion to a tough biological material, and to ultimately produce the natural squid sucker ring teeth.

Supramolecular β‐Sheet Suckerin–Based Underwater Adhesives

AbstractNature has evolved several molecular strategies to ensure adhesion in aqueous environments, where artificial adhesives typically fail. One recently‐unveiled molecular design for wet‐resistant adhesion is the cohesive cross‐β structure characteristic of amyloids, complementing the well‐established surface‐binding strategy of mussel adhesive proteins based on 3,4‐l‐dihydroxyphenylalanine (Dopa). Structural proteins that self‐assemble into cross β‐sheet networks are the suckerins discovered in the sucker ring teeth of squids. Here, light is shed on the wet adhesion of cross‐β motifs by producing recombinant suckerin‐12, naturally lacking Dopa, and investigating its wet adhesion properties. Surprisingly, the adhesion forces measured on mica reach 70 mN m−1, exceeding those measured for all mussel adhesive proteins to date. The pressure‐sensitive adhesion of artificial suckerins is largely governed by their cross‐β motif, as evidenced using control experiments with disrupted cross‐β domains that result in complete loss of adhesion. Dopa is also incorporated in suckerin‐12 using a residue‐specific incorporation strategy that replaces tyrosine with Dopa during expression in Escherichia coli. Although the replacement does not increase the long‐term adhesion, it contributes to the initial rapid contact and enhances the adsorption onto model oxide substrates. The findings suggest that suckerins with supramolecular cross‐β motifs are promising biopolymers for wet‐resistant adhesion.

A Short Peptide Hydrogel with High Stiffness Induced by 310-Helices to β-Sheet Transition in Water.

Biological gels generally require polymeric chains that produce long‐lived physical entanglements. Low molecular weight colloids offer an alternative to macromolecular gels, but often require ad‐hoc synthetic procedures. Here, a short biomimetic peptide composed of eight amino acid residues derived from squid sucker ring teeth proteins is demonstrated to form hydrogel in water without any cross‐linking agent or chemical modification and exhibits a stiffness on par with the stiffest peptide hydrogels. Combining solution and solid‐state NMR, circular dichroism, infrared spectroscopy, and X‐ray scattering, the peptide is shown to form a supramolecular, semiflexible gel assembled from unusual right‐handed 310‐helices stabilized in solution by π–π stacking. During gelation, the 310‐helices undergo conformational transition into antiparallel β‐sheets with formation of new interpeptide hydrophobic interactions, and molecular dynamic simulations corroborate stabilization by cross β‐sheet oligomerization. The current study broadens the range of secondary structures available to create supramolecular hydrogels, and introduces 310‐helices as transient building blocks for gelation via a 310‐to‐β‐sheet conformational transition.

Biological composites-complex structures for functional diversity.

The bulk of Earth's biological materials consist of few base substances-essentially proteins, polysaccharides, and minerals-that assemble into large varieties of structures. Multifunctionality arises naturally from this structural complexity: An example is the combination of rigidity and flexibility in protein-based teeth of the squid sucker ring. Other examples are time-delayed actuation in plant seed pods triggered by environmental signals, such as fire and water, and surface nanostructures that combine light manipulation with mechanical protection or water repellency. Bioinspired engineering transfers some of these structural principles into technically more relevant base materials to obtain new, often unexpected combinations of material properties. Less appreciated is the huge potential of using bioinspired structural complexity to avoid unnecessary chemical diversity, enabling easier recycling and, thus, a more sustainable materials economy.

Anion-Mediated Effects on the Size and Mechanical Properties of Enzymatically Crosslinked Suckerin Hydrogels.

The suckerin family of proteins, identified from the squid sucker ring teeth assembly, offers unique mechanical properties and potential advantages over other natural biomaterials. In this study, a small suckerin isoform, suckerin-12, is used to create enzymatically crosslinked, macro-scale hydrogels. Upon exposure to specific salt conditions, suckerin-12 hydrogels contracted into a condensed state where mechanical properties are found to be modulated by the salt anion present. The rate of contraction is found to correlate well with the kosmotropic arm of the Hofmeister anion series. However, the observed changes in hydrogel mechanical properties are better explained by the ability of the salt to neutralize charges in suckerin-12 by deprotonization or charge screening. Thus, by altering the anions in the condensing salt solution, it is possible to tune the mechanical properties of suckerin-12 hydrogels. The potential for suckerins to add new properties to materials based on naturally-derived proteins is highlighted.

Controlling Supramolecular Chiral Nanostructures by Self-Assembly of a Biomimetic β-Sheet-Rich Amyloidogenic Peptide.

Squid sucker ring teeth (SRT) have emerged as a promising protein-only, thermoplastic biopolymer with an increasing number of biomedical and engineering applications demonstrated in recent years. SRT is a supra-molecular network whereby a flexible, amorphous matrix is mechanically reinforced by nanoconfined β-sheets. The building blocks for the SRT network are a family of suckerin proteins that share a common block copolymer architecture consisting of amorphous domains intervened by smaller, β-sheet forming modules. Recent studies have identified the peptide A1H1 (peptide sequence AATAVSHTTHHA) as one of the most abundant β-sheet forming domains within the suckerin protein family. However, we still have little understanding of the assembly mechanisms by which the A1H1 peptide may assemble into its functional load-bearing domains. In this study, we conduct a detailed self-assembly study of A1H1 and show that the peptide undergoes β-strands-driven elongation into amyloid-like fibrils with a rich polymorphism. The nanostructure of the fibrils was elucidated by small and wide-angle X-ray scattering (SAXS and WAXS) and atomic force microscopy (AFM). The presence of His-rich and Ala-rich segments results in an amphiphilic behavior and drives its assembly into fibrillar supramolecular chiral aggregates with helical ribbon configuration in solution, with the His-rich region exposed to the solvent molecules. Upon increase in concentration, the fibrils undergo gel formation, while preserving the same mesoscopic features. This complex phase behavior suggests that the repeat peptide modules of suckerins may be manipulated beyond their native biological environment to produce a wider variety of self-assembled amyloid-like nanostructures.

Squid Suckerin Biomimetic Peptides Form Amyloid-like Crystals with Robust Mechanical Properties.

We present the self-assembly of fibers formed from a peptide sequence (A1H1) derived from suckerin proteins of squid sucker ring teeth (SRT). SRT are protein-only biopolymers with an unconventional set of physicochemical and mechanical properties including high elastic modulus coupled with thermoplastic behavior. We have identified a conserved peptide building block from suckerins that possess the ability to assemble into materials with similar mechanical properties as the native SRT. A1H1 displays amphiphilic characteristics and self-assembles from the bottom-up into mm-scale fibers initiated by the addition of a polar aprotic solvent. A1H1 fibers are thermally resistant up to 239 °C, coupled with an elastic modulus of ∼7.7 GPa, which can be explained by the tight packing of β-sheet-enriched crystalline building blocks as identified by wide-angle X-ray scattering (WAXS), with intersheet and interstrand distances of 5.37 and 4.38 Å, respectively. A compact packing of the peptides at their Ala-rich terminals within the fibers was confirmed from molecular dynamics simulations, and we propose a hierarchical model of fiber assembly of the mature peptide fiber.

Supramolecular β-Sheets Stabilized Protein Nanocarriers for Drug Delivery and Gene Transfection.

Suckerin proteins, recently discovered in the sucker ring teeth of squids, represent a family of promising structural biomacromolecules that can form supramolecular networks stabilized by nanoconfined β-sheets. Exploiting this feature as well as their specific amino acid composition, we demonstrate that artificial suckerin-19 (S-19) can be engineered into nanocarriers for efficient drug delivery and gene transfection in vitro and in vivo. First, we demonstrate that S-19 self-assembles into β-sheet stabilized nanoparticles with controlled particle sizes of 100-200 nm that are able to encapsulate hydrophobic drugs for pH-dependent release in vitro, and that can effectively inhibit tumor growth in vivo. We also show that S-19 can complex and stabilize plasmid DNA, with the complexes stabilized by hydrophobic interactions of the β-sheet domains as opposed to electrostatic interactions commonly achieved with cationic polymers, thus lowering cytotoxicity. The elevated Histidine content of S-19 appears critical to trigger endosomal escape by the proton sponge effect, thereby ensuring efficient gene transfection both in vitro and in vivo. Our study demonstrates that S-19 represents a promising functional protein nanocarrier that could be used for various drug and gene delivery applications.

Modular peptides from the thermoplastic squid sucker ring teeth form amyloid-like cross-β supramolecular networks.

The sucker ring teeth (SRT) located on the arms and tentacles of cephalopods represent as a very promising protein-based biopolymer with the potential to rival silk in biomedical and engineering applications. SRT are made of modular, block co-polymer like proteins (suckerins), which assemble into a semicrystalline polymer reinforced by nano-confined β-sheets, resulting in a supramolecular network with mechanical properties that match those of the strongest engineering polymers. In this study, we aimed to understand the molecular mechanisms behind SRT's self-assembly and robustness. The most abundant modular peptidic blocks of suckerin proteins were studied by various spectroscopic methods, which demonstrate that SRT peptides form amyloid-like cross-β structures.