Wheat germ agglutinin affinity chromatography enrichment and glyco-proteomic characterization of tetrodotoxin-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes).

Efficient enrichment of tetrodotoxin (TTX)-binding proteins from the plasma of cultured tiger pufferfish (Takifugu rubripes) was achieved by ammonium sulfate fractionation and wheat germ agglutinin (WGA) affinity chromatography. The enrichment efficiency was validated by ultrafiltration-LC/MS-based TTX-binding assay and proteomics. Major proteins in the WGA-bound fraction were identified as isoform X1 (125 kDa) and X2 variants (88 and 79 kDa) derived from pufferfish saxitoxin and tetrodotoxin-binding protein (PSTBP) 1-like gene (LOC101075943). The 125-kDa X1 protein was found to be a novel member of the lipocalin family, having three tandemly repeated domains. X2 variants, X2α and X2ß, were estimated to have two domains, and X2ß is structurally related to Takifugu pardalis PSTBP2 in their domain type and arrangement. Among 11 potential N-glycosylation sites in the X2 precursor, 5 N-glycosylated Asn residues (N55, N89, N244, N308, and N449) were empirically determined. Structural relationships among PSTBP homologs and complexity of their proteoforms are discussed.

Goals, challenges, and next steps in transdisciplinary fisheries research: perspectives and experiences from early-career researchers.

Fisheries are highly complex social-ecological systems that often face 'wicked' problems from unsustainable resource management to climate change. Addressing these challenges requires transdisciplinary approaches that integrate perspectives across scientific disciplines and knowledge systems. Despite widespread calls for transdisciplinary fisheries research (TFR), there are still limitations in personal and institutional capacity to conduct and support this work to the highest potential. The viewpoints of early career researchers (ECRs) in this field can illuminate challenges and promote systemic change within fisheries research. This paper presents the perspectives of ECRs from across the globe, gathered through a virtual workshop held during the 2021 World Fisheries Congress, on goals, challenges, and future potential for TFR. Big picture goals for TFR were guided by principles of co-production and included (i) integrating transdisciplinary thinking at all stages of the research process, (ii) ensuring that research is inclusive and equitable, (iii) co-creating knowledge that is credible, relevant, actionable, and impactful, and (iv) consistently communicating with partners. Institutional inertia, lack of recognition of the extra time and labour required for TFR, and lack of skill development opportunities were identified as three key barriers in conducting TFR. Several critical actions were identified to help ECRs, established researchers, and institutions reach these goals. We encourage ECRs to form peer-mentorship networks to guide each other along the way. We suggest that established researchers ensure consistent mentorship while also giving space to ECR voices. Actions for institutions include retooling education programs, developing and implementing new metrics of impact, and critically examining individualism and privilege in academia. We suggest that the opportunities and actions identified here, if widely embraced now, can enable research that addresses complex challenges facing fishery systems contributing to a healthier future for fish and humans alike.

Identification and Characterization of the Larval Settlement Pheromone Protein Components in Adult Shells of Crassostrea gigas: A Novel Function of Shell Matrix Proteins.

The global decline of natural oyster populations emphasizes the need to improve our understanding of their biology. Understanding the role of chemical cues from conspecifics on how oysters occupy appropriate substrata is crucial to learning about their evolution, population dynamics, and chemical communication. Here, a novel role of a macromolecular assembly of shell matrix proteins which act as Crassostrea gigas Settlement Pheromone Protein Components in adult shells is demonstrated as the biological cue responsible for gregarious settlement on conspecifics. A bioassay-guided fractionation approach aided by biochemical and molecular analyses reveals that Gigasin-6 isoform X1 and/or X2 isolated from adult shells is the major inducing cue for larval settlement and may also play a role in postlarva-larva settlement interactions. Other isolated Stains-all-stainable acidic proteins may function as a co-factor and a scaffold/structural framework for other matrix proteins to anchor within this assembly and provide protection. Notably, conspecific cue-mediated larval settlement induction in C. gigas presents a complex system that requires an interplay of different glycans, disulfide bonds, amino acid groups, and phosphorylation crosstalk for recognition. These results may find application in the development of oyster aquacultures which could help recover declining marine species and as targets of anti-fouling agents.

Regulatory Role of Sugars on the Settlement Inducing Activity of a Conspecific Cue in Pacific Oyster Crassostrea gigas.

This study evaluated the larval settlement inducing effect of sugars and a conspecific cue from adult shell extract of Crassostrea gigas. To understand how the presence of different chemical cues regulate settlement behavior, oyster larvae were exposed to 12 types of sugars, shell extract-coated and non-coated surfaces, and under varied sugar exposure times. Lectin-glycan interaction effects on settlement and its localization on oyster larval tissues were investigated. The results showed that the conspecific cue elicited a positive concentration dependent settlement inducing trend. Sugars in the absence of a conspecific cue, C. gigas adult shell extract, did not promote settlement. Whereas, in the presence of the cue, showed varied effects, most of which were found inhibitory at different concentrations. Sugar treated larvae exposed for 2 h showed significant settlement inhibition in the presence of a conspecific cue. Neu5Ac, as well as GlcNAc sugars, showed a similar interaction trend with wheat germ agglutinin (WGA) lectin. WGA-FITC conjugate showed positive binding on the foot, velum, and mantle when exposed to GlcNAc sugars. This study suggests that a WGA lectin-like receptor and its endogenous ligand are both found in the larval chemoreceptors and the shell Ethylenediaminetetraacetic acid (EDTA) extract that may complementarily work together to allow the oyster larva greater selectivity during site selection.