In Vitro Oxidative Crosslinking of Recombinant Barnacle Cyprid Cement Gland Proteins

Barnacle adhesion is a focus for fouling-control technologies as well as the development of bioinspired adhesives, although the mechanisms remain very poorly understood. The barnacle cypris larva is responsible for surface colonisation. Cyprids release cement from paired glands that contain proteins, carbohydrates and lipids, although further compositional details are scant. Several genes coding for cement gland-specific proteins were identified, but only one of these showed database homology. This was a lysyl oxidase-like protein (lcp_LOX). LOX-like enzymes have been previously identified in the proteome of adult barnacle cement secretory tissue. We attempted to produce recombinant LOX in E. coli, in order to identify its role in cyprid cement polymerisation. We also produced two other cement gland proteins (lcp3_36k_3B8 and lcp2_57k_2F5). lcp2_57k_2F5 contained 56 lysine residues and constituted a plausible substrate for LOX. While significant quantities of soluble lcp3_36k_3B8 and lcp2_57k_2F5 were produced in E. coli, production of stably soluble lcp_LOX failed. A commercially sourced human LOX catalysed the crosslinking of lcp2_57k_2F5 into putative dimers and trimers, and this reaction was inhibited by lcp3_36k_3B8. Inhibition of the lcp_LOX:lcp2_57k_2F5 reaction by lcp3_36k_3B8 appeared to be substrate specific, with no inhibitory effect on the oxidation of cadaverine by LOX. The results demonstrate a possible curing mechanism for barnacle cyprid cement and, thus, provide a basis for a more complete understanding of larval adhesion for targeted control of marine biofouling and adhesives for niche applications.

Description of Pallisentis thapari n. sp. and a re-description of Acanthosentis seenghalae (Acanthocephala, Quadrigyridae, Pallisentinae) using morphological and molecular data, with analysis on the validity of the sub-genera of Pallisentis

One new species of Pallisentis Van Cleave, 1928 is described from Channa punctatus (Bloch) from Gomti River (tributary of the Ganga River), in Uttar Pradesh, Lucknow district, India. Pallisentis thapari n. sp. is characterized morphologically by individuals having a globular proboscis armed with rooted hooks in 4 circles of 8–10 hooks each, the first circle more than 100 long and hooks gradually declining in size posteriorly. The trunk is cylindrical, with collar spines comprised of 15–17 complete circles of spines, each ring with 12–22 spines. As common in members of the genus, a narrow spine-free zone lies in between the rings of collar and trunk spines. Field of spines extends posteriorly to half of the body length, ending above the level of testes in males and slightly past mid-body in females; trunk spines have an optically-dense Y-shaped core. The trunk is only slightly wider at the anterior end. The syncytial cement gland of males contained 23–30 nuclei. Individuals of Channa striatus Bloch from the same locality also were infected with the new species. A second species of Acanthocephala, Acanthosentis seenghalae Chowhan, Gupta, Khera, 1988, was found as a parasite of Puntius sophore (Hamilton) from the same locality. The proboscis is short, globular, with 3 circles of hooks each circle bearing 6 hooks. The trunk is broad in the middle and tapered at both ends, with the posterior end narrower than the anterior end. Twelve to 16 circles of spines, each with 21–40 spines, extend from anterior end to just past mid-body in males and only to mid-body in females. The syncytial cement gland of males contains 6–10 nuclei. The analysis of 18s rDNA identified two clades of a monophyletic Pallisentis and placed the isolate of P. thapari n. sp. within that clade; previously established subgenera were not supported by the results of the analysis.

Insights into the Synthesis, Secretion and Curing of Barnacle Cyprid Adhesive via Transcriptomic and Proteomic Analyses of the Cement Gland

Barnacles represent one of the model organisms used for antifouling research, however, knowledge regarding the molecular mechanisms underlying barnacle cyprid cementation is relatively scarce. Here, RNA-seq was used to obtain the transcriptomes of the cement glands where adhesive is generated and the remaining carcasses of Megabalanus volcano cyprids. Comparative transcriptomic analysis identified 9060 differentially expressed genes, with 4383 upregulated in the cement glands. Four cement proteins, named Mvcp113k, Mvcp130k, Mvcp52k and Mvlcp1-122k, were detected in the cement glands. The salivary secretion pathway was significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the differentially expressed genes, implying that the secretion of cyprid adhesive might be analogous to that of saliva. Lysyl oxidase had a higher expression level in the cement glands and was speculated to function in the curing of cyprid adhesive. Furthermore, the KEGG enrichment analysis of the 352 proteins identified in the cement gland proteome partially confirmed the comparative transcriptomic results. These results present insights into the molecular mechanisms underlying the synthesis, secretion and curing of barnacle cyprid adhesive and provide potential molecular targets for the development of environmentally friendly antifouling compounds.

Ultrastructure of male Centrorhynchus globocaudatus (Acanthocephala) cement apparatus and function of cement gland secretion

Cement glands are one of the most conspicuous and distinctive elements of taxonomic interest in male Acanthocephala. Cement glands vary in shape, number and arrangement in different classes of the taxon. The glands and their products have a fundamental role in the reproductive process. Light and electron microscopy were used to investigate the ultrastructure of the cement apparatus, which includes both cement glands and the cement reservoir, in mature males of Centrorhynchus globocaudatus (Zeder, 1800). Centrorhynchus globocaudatus is an enteric parasite of birds of prey, including Falco tinnunculus (Linnaeus, 1758) and Buteo buteo (Linnaeus, 1758) from the province of Ferrara (northern Italy). The four elongated cement glands of C. globocaudatus are situated posterior to the testes. Sections through the cement glands show each gland is surrounded by a fibrous envelope with an approximate thickness of 0.6 μm. Beneath this envelope is an outer cytoplasmic layer thickness ranging from 22 to 26 μm, which contains a number of nuclei with diameters variable from 20 to 22 μm. The cytoplasmic layer is filled with prominent free ribosomes and many mitochondria with lamellar cristae. Secretory granules, measuring from 1 to 1.3 μm in diameter, are formed within the cytoplasmic layer. The cytoplasmic layer surrounds the luminal area for storage of the cement material in each gland. Cement gland ducts arise from the gland and extend towards a common cement reservoir in close contact with the seminal vesicle and Saefftigen's pouch. Microtubules, large secretory granules and rest of undefined organelles were also observed within the cement reservoir.

Identification and Description of the Key Molecular Components of the Egg Strings of the Salmon Louse (Lepeophtheirus salmonis)

The salmon louse Lepeophtheirus salmonis is a parasite of Atlantic salmon and other salmonids. Every year, it causes high costs for the Norwegian aquaculture industry. While the morphology of the female genital tract has been described, knowledge of the molecular basis of reproduction is very limited. We identified nine genes which are expressed exclusively in the female cement gland, the organ responsible for cement production, which is used to hold the eggs together and keep them attached to their mother in egg strings. Six of these genes encode proteins with signal peptides and probably form the main component of the cement. Two other genes are peroxidases, which are probably important in the cement formation. The last gene is not similar to any known protein, but contains a transmembrane domain. A knockdown of all these genes leads to missing or deformed egg strings, preventing reproduction of the lice. The correct assemblage of the cement in the cement gland is essential for successful reproduction of salmon lice. Similar proteins seem to be present in other copepod species, as well.

Decrease in size at maturity of female American lobsters Homarus americanus (H. Milne Edwards, 1837) (Decapoda: Astacidea: Nephropidae) over a 50-year period in Maine, USA

ABSTRACTThe size at which female American lobsters Homarus americanus (H. Milne Edwards, 1837) reach maturity was determined for females collected from inshore (< 3 nautical miles, or 5.5 km) Boothbay Harbor (BBH), Maine, USA in 2018. A total of 272 females were collected during a three-week period in the spring and each female was assigned a maturity status (immature or mature) based on ovarian staging. These determinations were then compared to two similar female-maturity studies undertaken in BBH by the Maine Department of Marine Resources over the last 50 years. The comparison revealed that the length at which 50% of females reach maturity has decreased by 5 mm over the last 25 years and a significant difference between maturity ogives generated over time in BBH. Cement-gland stage was also recorded for all females and further analysis revealed no significant difference between maturity ogives generated using ovarian and cement-gland staging. Results indicate that cement-gland staging is an effective maturity assessment method in female lobsters from this region. Because there is a well-documented relationship between temperature and size at maturity in crustaceans, we also examined changes in sea-surface temperature during this period in BBH. We found that the region has warmed significantly over this period of time with extreme high temperatures occurring more frequently during the last 25 years. Our results can be used to update key parameters in the stock-assessment model related to growth and egg production in the Gulf of Maine/George’s Bank stock. These findings can also be used to inform future management decisions related to the carapace length of harvested lobsters and the preservation of mature females.

Patterning of the vertebrate head in time and space by BMP signalling

How head patterning is regulated in vertebrates is yet to be understood. In this study, we show that frog embryos injected with Noggin at different blastula and gastrula stages had their head development sequentially arrested at different positions. When timed BMP inhibition was applied to BMP-overexpressing embryos, the expression of five genes: xcg-1 (a marker of the cement gland, which is the front-most structure in the frog embryo), six3 (a forebrain marker), otx2 (a forebrain and mid-brain marker), gbx2 (an anterior hindbrain marker) and hoxd1 (a posterior hindbrain marker) were sequentially fixed. These results suggest that timed interactions between BMP and anti-BMP are involved in patterning the vertebrate head progressively in time and space. Since the above genes are not expressed sequentially, there may be a BMP dependent gene sequence during head patterning that can be arrested by BMP inhibition and regulate the specification of positional values in the head.