Operculum ultrastructure in leech cocoons

Clitellate annelids (i.e., oligochaetes including leeches) secrete cocoons as part of their normal reproductive cycle. Typically, the cocoon sheath is passed over the head of the leech and sealed at both ends by opercula (i.e., glue‐like material secreted by the clitellum). Both the fibrous cocoon wall (CW) and opercula are chemically‐related biomaterials that share unusual physiochemical properties, including thermal and chemical resiliency. To explore the underlying morphology of the operculum, we examined cocoons from four leech species (i.e., Myzobdella lugubris, Theromyzon tessulatum, Erpobdella obscura, and Erpobdella punctata) by transmission (TEM) and scanning electron microscopy (SEM). Transmission electron micrographs of all opercula revealed a common, ultrastructural pattern comprising an electron‐dense mosaic of ordered polygons that surrounded interspersed cavities. The long axes of cavities were often oriented directionally, suggesting that operculum material is pliable prior to solidification and distorted as a consequence of cocoon deposition. Concomitantly, the operculum permeates jagged edges of the cocoon sheath sealing the cocoon, which provides a mechanically strong CW/operculum boundary. SEM of leech opercula revealed globular nanoparticles comparable to that observed in bioadhesives from disparate animal phyla (e.g., mussel, barnacle, sea star), suggesting a convergent mechanism of bioadhesion among animals. J. Morphol. 274:940–946, 2013. © 2013 Wiley Periodicals, Inc.     

Structural model of an antistasin/notch-like fusion protein from the cocoon wall of the aquatic leech, Theromyzon tessulatum

The aquatic leech, Theromyzon tessulatum, secretes a proteinaceous cocoon with extraordinary physical properties (e.g., proteolytic, thermal resiliency). The deduced amino acid sequence of a major protein (Tcp—Theromyzon cocoon protein) from the T. tessulatum cocoon wall has been used to model the endogenous structure of the Tcp protein. The Tcp protein sequence comprises six internal repeats, each containing 12 ordered Cys residues. Amino acid alignments suggest that the region Cys1→6 is homologous to antistasin, a leech anticoagulant, and Cys7→12 is homologous to an epidermal growth factor-like domain found in notch-class proteins, which play critical roles in development, signaling, and adhesion throughout the Animalia. Modeling of individual domains (i.e., antistasin and notch) positions multiple hydrophobic and charged residues on the surface. When the antistasin and notch domains were fused, hydrophobic pockets appeared that may facilitate a polymerization mechanism. Collectively, the predicted features of our Tcp model are consistent with the physical properties of the leech cocoon wall.      

Cell dynamics during cocoon secretion in the aquatic leech, Theromyzon tessulatum (Annelida: Clitellata: Glossiphoniidae)

One distinguishing feature of clitellate annelids is the presence of specialized segments comprising the clitellum, whose primary function is to secrete a cocoon. Using histological analyses, we have documented cell types (I-V) and cellular processes associated with cocoon secretion in the aquatic leech, Theromyzon tessulatum. Our data indicate that the bulk of the cocoon's biomass arises from precursor cells of a single type that hypertrophy and proliferate ∼1 week prior to egg laying, and then differentiate into either of two cell types (i.e., Type II or Type III) depending on their position within the clitellum. Type II cells are concentrated along the lateral edges and venter of the clitellum and secrete alcian blue-staining granules that form opercula (i.e., glue-like material that seals both cocoon ends), while Type III cells populate the dorsal midline and secrete azocarmine-staining granules that build the cocoon wall. Both cell types occupy spaces between deep muscle layers and extend long-neck tubules to the surface epithelium as they fill with granules a few days prior to egg laying. Other cell types appear to make minor contributions to the cocoon (e.g., Type I, Type IV) or have supporting or signaling roles (e.g., Type V). Our observations suggest that post-translational modification (i.e., glycosylation) of the same core protein(s) distinguishes the granules of Type II/III cells, and that the default state of the Type II/III precursor may be evolutionarily linked to secretory cells in basal polychaetes.     

Operculum regeneration following failed predation in the Silurian gastropod Oriostoma

Regeneration of the calcareous rigiclaudent operculum following severe, non‐lethal, predatory attacks is described in two specimens of the characteristic Silurian gastropod Oriostoma from the English Midlands. This is the first record of the regeneration of opercula in Palaeozoic gastropods and of opercula conjoined with shells that have been repaired after significant, failed, durophagous attacks. In one specimen, a series of repaired injuries culminates in a repaired aperture closed by the operculum. In the second specimen, new shell growth has failed to recover the full extent of the original broken whorl, and the new aperture and operculum, though well developed, are smaller than the originals. Both opercula are unusually thin centrally when compared to other, strongly domed, Oriostoma opercula.     

Cretaceous and living colloniidae of the redefined subfamily Petropomatinae,with two new genera and one new species,with notes on opercular evolution in turbinoideans,and the fossil record of Liotiidae (Vetigastropoda: Turbinoidea)

Opercular characters are of major importance to understanding the living and fossil groups in the vetigastropod superfamily TurbinoideaRafinesque. Here we evaluate current knowledge of fossil turbinoid opercula in the light of a newly discovered Recent colloniid with an unfamiliar opercular morphology. The operculum ofLiotipoma n. gen. has a multispiral and conical inner surface; the outer surface is rugose with a central pit. Similar opercula are known from the Late CretaceousSohlipoma n. gen. and the Early CretaceousPetropoma Gabb. These genera are assigned to the here redefined colloniid subfamily PetropomatinaeCox inKnight et al., which was based originally onGabb’s misinterpretation of the inner and outer sides of the operculum ofPetropoma peruana Gabb. In contrast, opercula of most living members of Colloniinae s.S., have a flat inner side with a multispiral pattern that becomes paucispiral. In addition, opercula typical for living Turbinidae are here traced to the Late Cretaceous. The new genusLiotipoma has lamellar sculpture like that of Liotiidae H. & A.Adams, showing that such sculpture is not restricted to the Liotiidae. We also reject previous allocations of the Permian DichostasiinaeYochelson and Jurassic Cross-ostomatinaeCox inKnight et al. to Liotiidae, and find an undisputed earliest known Jurassic occurrence for the Liotiidae. The recent suggestion that the change from corneous to calcareous opercula among turbinids occurred concurrently with a change in pattern from multispiral to paucispiral is not supported by our data.     

Type IX secretion: the generation of bacterial cell surface coatings involved in virulence,gliding motility and the degradation of complex biopolymers

The Type IX secretion system (T9SS) is present in over 1000 sequenced species/strains of the Fibrobacteres‐Chlorobi‐Bacteroidetes superphylum. Proteins secreted by the T9SS have an N‐terminal signal peptide for translocation across the inner membrane via the SEC translocon and a C‐terminal signal for secretion across the outer membrane via the T9SS. Nineteen protein components of the T9SS have been identified including three, SigP, PorX and PorY that are involved in regulation. The inner membrane proteins PorL and PorM and the outer membrane proteins PorK and PorN interact and a complex comprising PorK and PorN forms a large ring structure of 50 nm in diameter. PorU, PorV, PorQ and PorZ form an attachment complex on the cell surface of the oral pathogen, Porphyromonas gingivalis. P. gingivalis T9SS substrates bind to PorV suggesting that after translocation PorV functions as a shuttle protein to deliver T9SS substrates to the attachment complex. The PorU component of the attachment complex is a novel Gram negative sortase which catalyses the cleavage of the C‐terminal signal and conjugation of the protein substrates to lipopolysaccharide, anchoring them to the cell surface. This review presents an overview of the T9SS focusing on the function of T9SS substrates and machinery components.     

The external gills of anuran amphibians: Comparative morphology and ultrastructure

The external gills of anuran amphibians are transient structures, covered by the development of the operculum and regressing soon afterwards. Their functional role has been regarded as equivocal. However, detailed morphological analysis has been limited. Analysis of 21 species from six families using scanning and transmission electron microscopy revealed diversity at the anatomical and cellular levels in extent and length of gill filaments, numbers of surface ciliated cells, width of water‐blood barrier distance, and evidence of gill motility. The most highly developed external gills were found in species with delayed hatching, such as Phyllomedusa trinitatis, or in species in which hatchlings hang from the surface film of temporary ponds, such as Phrynohyas venulosa in which gills added 26–38% to body surface area. In one family, the bufonids, all four species examined had poorly developed gills, but in other families where we examined several species, the hylids and leptodactylids, there was considerable diversity of external gills, suggesting flexible adaptation to incubation and hatching environment. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

The ultrastructure of malpighian tubules and the chemical composition of the cocoon of Aeolothrips intermedius Bagnall (Thysanoptera)

The secretory activity of the two branched malpighian tubules (MTs) of the second‐instar larva in Aeolothrips intermedius is described. MTs of adult thrips have the typical ultrastructure of excretory epithelium with apical microvilli containing long mitochondria and a rich system of basal membrane infoldings. In the second‐instar larva just before pupation, the ultrastructure of MT epithelial cells is dramatically different, and there are numerous huge Golgi systems in the cytoplasm. These cells are involved in an intense secretory activity to produce an electron‐dense product which is released into the MTs lumen. This secretion is extruded from the hindgut and used by the second‐instar larva to build an elaborate protective cocoon for pupation. Electron‐spray‐ionization mass spectrometry analysis of the cocoon revealed the presence of a β‐N‐acetyl‐glucosamine, the main component of chitin, which is also present in the cocoons of Neuroptera and some Coleoptera. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of starvation‐induced dispersion in Pseudomonas putida biofilms: genetic elements and molecular mechanisms

Pseudomonas putida OUS82 biofilm dispersal was previously shown to be dependent on the gene PP0164 (here designated lapG). Sequence and structural analysis has suggested that the LapG geneproduct belongs to a family of cysteine proteinases that function in the modification of bacterial surface proteins. We provide evidence that LapG is involved in P. putida OUS82 biofilm dispersal through modification of the outer membrane‐associated protein LapA. While the P. putida lapG mutant formed more biofilm than the wild‐type, P. putida lapA and P. putida lapAG mutants displayed decreased surface adhesion and were deficient in subsequent biofilm formation, suggesting that LapG affects LapA, and that the LapA protein functions both as a surface adhesin and as a biofilm matrix component. Lowering of the intracellular c‐di‐GMP level via induction of an EAL domain protein led to dispersal of P. putida wild‐type biofilm but did not disperse P. putida lapG biofilm, indicating that LapG exerts its activity on LapA in response to a decrease in the intracellular c‐di‐GMP level. In addition, evidence is provided that associated to LapA a cellulase‐degradable exopolysaccharide is part of the P. putida biofilm matrix.     

Crystal structure of the C‐terminal domain of the Salmonella type III secretion system export apparatus protein InvA

InvA is a prominent inner‐membrane component of the Salmonella type III secretion system (T3SS) apparatus, which is responsible for regulating virulence protein export in pathogenic bacteria. InvA is made up of an N‐terminal integral membrane domain and a C‐terminal cytoplasmic domain that is proposed to form part of a docking platform for the soluble export apparatus proteins notably the T3SS ATPase InvC. Here, we report the novel crystal structure of the C‐terminal domain of Salmonella InvA which shows a compact structure composed of four subdomains. The overall structure is unique although the first and second subdomains exhibit structural similarity to the peripheral stalk of the A/V‐type ATPase and a ring building motif found in other T3SS proteins respectively.     

Trypanosoma cruzi: Identification of DNA targets of the nuclear periphery coiled-coil protein TcNUP-1

The nuclear lamina is a structure that lines the inner nuclear membrane. In metazoans, lamins are the primary structural components of the nuclear lamina and are involved in several processes. Eukaryotes that lack lamins have distinct proteins with homologous functions. Some years ago, a coiled-coil protein in Trypanosoma brucei, NUP-1, was identified as the major filamentous component of its nuclear lamina. However, its precise role has not been determined. We characterized a homologous protein in Trypanosoma cruzi, TcNUP-1, and identified its in vivo DNA binding sites using a chromatin immunoprecipitation assay. We demonstrate for the first time that TcNUP-1 associates with chromosomal regions containing large non-tandem arrays of genes encoding surface proteins. We therefore suggest that TcNUP-1 is a structural protein that plays an essential role in nuclear organization by anchoring T. cruzi chromosomes to the nuclear envelope.     

The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein

Colicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cell's outer membrane barrier to kill. To achieve this, most colicins require an abundant porin (e.g. OmpF) plus a low‐copy‐number, high‐affinity, outer membrane protein receptor (e.g. BtuB). Recently, genetic screens have suggested that colicin N (ColN), which has no high‐affinity receptor, targets highly abundant lipopolysaccharide (LPS) instead. Here we reveal the details of this interaction and demonstrate that the ColN receptor‐binding domain (ColN‐R) binds to a specific region of LPS close to the membrane surface. Data from in vitro studies using calorimetry and both liquid‐ and solid‐state NMR reveal the interactions behind the in vivo requirement for a defined oligosaccharide region of LPS. Delipidated LPS (LPS^ΔLIPID) shows weaker binding; and thus full affinity requires the lipid component. The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF. ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.     

NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip‐translocon protein–protein interactions

The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein–protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N‐terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/β domain and a portion of its coiled‐coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N‐terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip‐translocon protein–protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097–1107. © 2016 Wiley Periodicals, Inc.     

The Glycerol‐3‐Phosphate Acyltransferase TbGAT is Dispensable for Viability and the Synthesis of Glycerolipids in Trypanosoma brucei

Glycerolipids are the main constituents of biological membranes in Trypanosoma brucei, which causes sleeping sickness in humans. Importantly, they occur as a structural component of the glycosylphosphatidylinositol lipid anchor of the abundant cell surface glycoproteins procyclin in procyclic forms and variant surface glycoprotein in bloodstream form, that play crucial roles for the development of the parasite in the insect vector and the mammalian host, respectively. The present work reports the characterization of the glycerol‐3‐phosphate acyltransferase TbGAT that initiates the biosynthesis of ester glycerolipids. TbGAT restored glycerol‐3‐phosphate acyltransferase activity when expressed in a Leishmania major deletion strain lacking this activity and exhibited preference for medium length, unsaturated fatty acyl‐CoAs. TbGAT localized to the endoplasmic reticulum membrane with its N‐terminal domain facing the cytosol. Despite that a TbGAT null mutant in T. brucei procyclic forms lacked glycerol‐3‐phosphate acyltransferase activity, it remained viable and exhibited similar growth rate as the wild type. TbGAT was dispensable for the biosynthesis of phosphatidylcholine, phosphatidylinositol, phosphatidylserine, and GPI‐anchored protein procyclin. However, the null mutant exhibited a slight decrease in phosphatidylethanolamine biosynthesis that was compensated with a modest increase in production of ether phosphatidylcholine. Our data suggest that an alternative initial acyltransferase takes over TbGAT's function in its absence.     

Ultrastructural study on cocoon formation in the freshwater oligochaete,Tubifex hattai

The clitellar epithelium of the freshwater oligochaete, Tubifex hattai, is composed of four types of gland cells (Type I, II, III, and IV), in addition to the cells generally found in the epidermis of this worm. The possible function of these gland cells in cocoon formation was studied with the electron microscope. Type I cells discharge their secretory granules by means of compound exocytosis and provide the materials for the future cocoon membrane. Immediately after completion of the discharge from Type I cells, Type II and III cells simultaneously discharge their secretory granules by means of compound exocytosis. The secretions from Type II cells constitute a colloid in the cocoon lumen and probably cause structural modifications in the future cocoon membrane. The secretory products from Type III cells form the cocoon plug. Although the process of discharge of secretory granules from Type IV cells was not observed, the contribution of these cells to the cocoon formation, producing hoops on the outer surface of the future cocoon membrane and fixing its anterior ends on the clitellum, is inferred from a morphological comparison of the hoop and the structure of the secretory granules.     

Comparing aging precision of calcified structures in shovelnose sturgeon

Age estimates for population analysis must be precise. We assessed the usefulness of pectoral fin rays, sphenoids, opercula, and dorsal scutes of shovelnose sturgeonScaphirhynchus platorynchus (n = 30) as aging structures based on ease of collection, distinctness of annuli, and measures of precision both between and within readers. We also determined how age estimates from paired fin rays of individuals were related (n = 106). Pectoral fin rays generated the highest within‐reader precision (100% within 2 years) followed by sphenoids (58%), opercula (56%), and dorsal scutes (49%). Ages estimated by the pectoral fin ray also had higher between‐reader agreement (80% within 1 year) than did those from the operculum (60%), sphenoid (59%), or dorsal scute (56%). Likewise, age estimates from pectoral fin rays had the lowest mean coefficient of variation (8.2%) followed by sphenoids (9.9%), opercula (11.3%), and dorsal scutes (11.5%). Only the operculum produced biased estimates between readers. Ages from paired fin rays agreed poorly (36% exact, 30% within 1 year) although no aging bias occurred. The pectoral fin ray is typically used to age shovelnose sturgeon. Because uncertainty about accuracy and precision of age estimates from this structure remains, shovelnose sturgeon management objectives that result from age data should remain conservative.     

Of mudsnails,terrapins, and flukes: Use of trematodes as a field‐based project in parasitology research

We present an exercise for counting trematode cysts on mudsnails that can be implemented as a field‐based project in course work by students or by volunteers. The exercise involves the digenetic trematode Pleurogonius malaclemys, which infects diamondback terrapins (Malaclemys terrapin) as its definitive host, and eastern mudsnails (Tritia obsoleta) as its intermediate host. The trematode forms macroscopic metacercarial cysts on the shells and opercula of the mudsnails, and the life cycle is completed when terrapins ingest the intermediate hosts with cysts. Previous research has suggested that quantifying cysts on mudsnails can be a relatively simple method to estimate terrapin population sizes where terrapins co‐occur with mudsnails. Three non‐professional scientist groups (including students and volunteers) were tested in their estimates of prevalence and intensity data compared to verified cyst counts. Prevalence data were relatively accurate (~76–78%) and reliable (68–89%) across the groups. The intensity of cysts (on shells and opercula of mudsnails) were mostly within 12% of the verified counts (total mean intensity of 1.81–2.97 cysts/parasitized mudsnail) but varied significantly in two cases due to false positives (likely due to sand grains or debris scored as cysts) or negatives (likely due to missed cysts behind the operculum). The exercise can be completed as a lab or series of labs in courses, and data from such projects could be used in terrapin conservation efforts, particularly in helping scientists determine where terrapins occur. We discuss modification of the exercise for other trematode species in regions around the world and hope this expands recognition by the public of the important role of parasites in ecosystems.     

The Toxoplasma gondii rhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

In mice, avirulent strains (e.g. types II and III) of the protozoan parasite Toxoplasma gondii are restricted by the immunity‐related GTPase (IRG) resistance system. Loading of IRG proteins onto the parasitophorous vacuolar membrane (PVM) is required for vacuolar rupture resulting in parasite clearance. In virulent strain (e.g. type I) infections, polymorphic effector proteins ROP5 and ROP18 cooperate to phosphorylate and thereby inactivate mouse IRG proteins to preserve PVM integrity. In this study, we confirmed the dense granule protein GRA7 as an additional component of the ROP5/ROP18 kinase complex and identified GRA7 association with the PVM by direct binding to ROP5. The absence of GRA7 results in reduced phosphorylation of Irga6 correlated with increased vacuolar IRG protein amounts and attenuated virulence. Earlier work identified additional IRG proteins as targets of T. gondii ROP18 kinase. We show that the only specific target of ROP18 among IRG proteins is in fact Irga6. Similarly, we demonstrate that GRA7 is strictly an Irga6‐specific virulence effector. This identifies T. gondii GRA7 as a regulator for ROP18‐specific inactivation of Irga6. The structural diversity of the IRG proteins implies that certain family members constitute additional specific targets for other yet unknown T. gondii virulence effectors.     

Mating behavior,spawning, parental care,and embryonic development of some marine pseudocerotid flatworms (Platyhelminthes: Rhabditophora: Polycladida) in Singapore

The reproductive biology of tropical marine cotylean polyclads is presently poorly known. Reproductive aspects of 16 polyclad species from the family Pseudocerotidae in the genera Acanthozoon, Nymphozoon, Phrikoceros, Pseudobiceros, Pseudoceros, Thysanozoon, and Tytthosoceros from Singapore were documented for the first time. Penis fencing was observed to be just a mating ritual and not necessary for insemination, not always aggressive, and could also result in eventual reciprocal insemination. Results showed that all species underwent similar embryonic developmental stages and hatched as Müller's larvae. Only Pseudoceros concinnus and Pseudoceros laingensis, with mean developmental times of >20 days and mean brood sizes of <1,000 eggs, displayed long‐term parental care. Polyclads producing larger brood sizes had shorter developmental times and only covered their egg masses for about one day. Phrikoceros baibaiye and Pseudobiceros spp. produced egg capsules with pointed opercula, whereas all other species possessed smooth, rounded opercula. All genera hatched with brownish‐orange larvae, except for Pseudoceros spp., which hatched with reddish‐purple larvae regardless of the initial embryo color (either grayish‐yellow or reddish‐purple). These could potentially complement current taxonomic characters in distinguishing polyclad genera and species.