Introduction
Limiting the spread of classical and emergent microbial infections in aquaculture is a challenge the scientific community is increasingly aware of, looking for new efficient preventive and therapeutic approaches. Several synthetic antimicrobial agents have been used to control bacterial infections in shellfish and fish cultured worldwide. However, around 90% of aquatic bacteria are resistant to at least one antibiotic, threatening human health shortly (Elbashir et al, 2018). Marine species compose around half of the total global biodiversity, and considering their unique living environment (composition, close contact with microbes, and properties, e.g., antibacterial, antiviral, antitumoral), these organisms have gained substantial importance as a “gold mine” for the search of new natural bioactive compounds (Cheung et al. 2015). Therefore, in the BIOPTAL “Bioactive Octopus peptides with potential for aquaculture” Marie Skłodowska-Curie project (https://cordis.europa.eu/project/id/101026577), we are focused on high-throughput protein search from an under-explored marine reservoir of peptide diversity – the common octopus (Octopus vulgaris). O. vulgaris is one of the most demanded cephalopod species for human consumption and represents a challenge for aquaculture farming. Octopuses routinely edit their RNA sequences to adapt to their environment (Alon et al, 2015), meaning that some pertinent bioactive compounds with antimicrobial or other relevant properties to several industries, such as aquaculture, must only occur under particular environmental conditions. Unraveling putative bioactive compounds from O. vulgaris can help develop novel therapeutic solutions for several marine species to fight the appearance and persistence of multidrug-resistant bacterial strains (e.g., the pathogenic gram-negative bacterium, Vibrio parahaemolyticus, found in marine and estuarine environments which causes a varying degree of illness including gastroenteritis) in the aquaculture sector. Thus, aiming to detect natural compounds highly expressed under challenging conditions and stimulate bioactive peptide secretion to produce different kinds of compounds involved in host defense mechanisms, we accomplished an in vivo bacterial challenge with V. parahaemolyticus and generated the O. vulgaris proteome to investigate the differential expression of several antimicrobial and toxins-related genes in the skin, posterior salivary glands (PSGs), and hemocytes of the common octopus.
Materials and methods
Ten adult specimens of common octopus were obtained from the Mediterranean Sea (Valencia, Spain), maintained in the Marine Fish Facility at the University of Murcia (Spain), and randomly distributed into two groups: i) control group (unchallenged, immersion with sterile sea water) and ii) bath-challenged with V. parahaemolyticus (challenged, immersion with a sub-lethal bacteria concentration). After 6 h bath-challenged with the bacteria, octopuses were humanely sacrificed (Fiorito et al, 2015) and dissected. Hemolymph, skin, and PSGs fragments were collected to obtain the proteome of these tissues and hemocyte cells. Digestion of the samples was performed with trypsin/LysC overnight following SP3 – solid-phase-preparation (Hughes et al, 2019), and the concentration of the resulting peptides was measured by fluorescence. The shotgun proteomics analysis of the two tissues and cells was performed by injection of peptides in a nano-LC (Ultimate 3000, Thermo Fisher Scientific, Bremen, Germany) connected to a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The LC-MS/MS raw data were analyzed by Proteome Discoverer SP1 3.0 software (Thermo Fisher Scientific) for protein identification against the following databases UniProtKB/Swiss-Prot, UniProtKB/Swiss-Prot Tox-Prot, Starpep (Aguilera-Mendoza et al, 2019), and a comprehensive non-redundant antimicrobial peptides (AMPs) database (Almeida et al, 2020) together with a database of common contaminants from MaxQuant.
Results and discussion
AMPs present great diversity considering their structure, activity, mode of action, and genetic origin (Almeida et al, 2020). These peptides are widely distributed among prokaryotes, animals, and plants, establishing the first line of defense against microorganisms (bacteria, viruses, parasites, and fungi) as part of their primary immune system. A previous proteomics study of unchallenged O. vulgaris from eastern Atlantic waters (Portuguese waters) suggests the putative production of AMPs by O. vugaris PSGs as part of their primary immune system (Almeida et al, 2020). That study revealed that the O. vulgaris PSGs antimicrobial repertoire is composed of AMPs found in relatively high abundance, such as ubiquitin-derived peptides (cgUbiquitin), histone-derived peptides (buforin-II, H2A, and H2B), peptides similar to bovine pancreatic trypsin inhibitor (BPTI/Magainin), among others (Almeida et al, 2020). In this project, we are implementing a high-throughput approach for discovering new antimicrobial and immunomodulatory peptides potentially expressed under challenge (V. parahaemolyticus) from tissues and cells of O. vulgaris. We are looking specifically for differential expression of the previously mentioned AMPs and other AMPs not yet characterized in this species. Our results (work in progress) will help shed light on the underexplored antimicrobial repertoire of the common octopus, uncovering potential new peptides and simultaneously elucidating the role of glands, skin, and hemocytes in the species’ molecular environmental adaptation, which remains poorly known (Fingerhut et al, 2018), opening the opportunity to develop alternatives to fight the synthetic antimicrobial resistance problem currently impacting several industrial sectors such as aquaculture.
Conclusion
This study offers a comprehensive proteomic view to enlarge the understanding of the common octopus’s adaptive mechanisms, broadening the knowledge on the first line of defense against multi-antibiotic resistant bacteria affecting the aquaculture sector. The information provided by this study represents a pilot prospect that could help optimize culture conditions and disease prevention procedures affecting aquaculture-relevant species.
Acknowledgments
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-a-Curie grant agreement No 101026577 (D.A.).
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