Introduction
Animal welfare is a growing public concern, especially for the emerging field of octopus aquaculture. The common octopus, Octopus vulgaris, is a commercial valuable species, and an excellent candidate for aquaculture diversification (1). Overfishing of nat ural stocks and high market demand make sustainable octopus aquaculture essential to reduce fishing pressure and protect natural populations. To achieve this, optimizing and standardizing practices to control and safeguard animal welfare is crucial. Welfare assessment should prioritize aspects such as diet, appropriate housing conditions, environmental enrichment, behavior, and health. In this context, the early detection of pathogens, particularly the protozoan parasite Aggregata octopiana and bacteria of the Vibrio genus, is essential to prevent disease outbreaks and reduce animal suffering (2-3). Moreover, the identification of protein- or microbiota- based welfare markers from non-invasive samples, such as skin mucus, could provide powerful tools for routine health monitoring, offering insight into the physiological status of the animals without compromising their welfare. This study identifies specific octopus welfare markers, defined as biological Operational Welfare Indicators (OWIs), to be integrated into monitoring plans for good aquaculture practices.
Material and methods
Quantitative PCR (qPCR) assays were developed for the absolute quantification of the coccidian A. octopiana in fecal samples, using standard curves based on a plasmid-cloned target 18S rDNA sequence with known copy numbers. qPCR results were correlated with microscopic examination of fecal smears, applying an optimized algorithm in Python 3.11.8 that selected t hreshold pairs from qPCR Ct values and microscopy count, allowing the establishment of different infection ranges of infection. A similar qPCR approach was used to quantify Vibrio spp. from tank seawater and skin mucus samples, employing genus-specific primers, and results were validated through colony counts on selective TCBS agar. A shotgun proteomics approach combined with liquid chromatography coupled with tandem mass spectrometry (LC-ESI-MS/MS) using an Orbitrap-Elite instrument was used to create a reference dataset from octopus skin mucus including wild and aquarium maintained octopus, as well as adult and senescent ones . The final proteome compilation was investigated by integrated in silico studies, including Gene Ontology (GO), the Kyoto Encyclope dia of Genes and Genomes (KEGG) pathway, network studies, and prediction and characterization analysis of potential bioactive peptides. Additionally, tank seawater and skin samples from individuals showing external lesions were subjected to shotgun metagenomics sequencing (RNA-seq Illumina) to analyze microbial community structure and potential pathogens associated with compromised skin integrity. All samples were collected from octopuses under aquaculture conditions.
Results
Absolute quantifications of A.octopiana and Vibrio spp by qPCR enabled rapid, reliable, and low-impact monitoring of infection in aquarium-maintained octopuses, supporting early diagnosis and improved animal welfare. The quantification of A. octopiana 18S rDNA by qPCR using plasmid-based standard curves achieved a high efficiency (97%, R² = 0.995) and a detection limit of 10² copies/μL . The assay showed strong repeatability (CV = 0.01) and high specificity . The comparison between data obtained by qPCR and those obtained with microscopy counts using an optimized algorithm let to define three infection levels (low, medium, high), yielding a good corre lation (r = 0.723) between both methods. This allowed reliable, quantitative categorization of parasite load in non-invasively collected samples. In addition, absolute quantification of Vibrio spp. was successfully implemented using genus-conserved primers in a qPCR assay. The correlation between Ct values obtained by qPCR and colony counts on selective TCBS agar validated the method and enabled the identification of potentially pathogenic Vibrio colonies in culture conditions. Proteomic analysis revealed a different pattern between conditions. Although similar profiles were observed, differences in relative abundances led to the identification of potential biomarkers, including caspase-3-like, protocadherin 4, deleted in malignant brain tumors, thioredox in, papilin, annexin, cofilin and mucin-4 proteins. Some of these proteins also revealed potential as bioactive peptides , with several displaying antibacterial and antiviral activities in skin mucus, as identified using the CAMP (Collection of Anti-Microbial Peptides) database. The metagenomics analysis identified different bacterial species corresponding mainly to the genus Vibrio as causative of skin ulcerations .
Discussion
Ensuring animal welfare in aquaculture systems is essential, and the identification of reliable OWIs is key to achieving this goal. Although octopus farming is not yet established at an industrial scale, preserving welfare in aquarium-maintained individuals is crucial, both for ethical husbandry and as a base for future large-scale aquaculture practices. This study highlights the effectiveness of non-invasive molecular tools for monitoring the health and welfare of O. vulgaris under aquarium conditions. The validated qPCR assays for A. octopiana and Vibrio spp. enabled sensitive, repeatable, and early detection of infections, supporting their use as practical OWIs. The strong correlation between molecular and traditional diagnostic methods further reinforces their reliability.
Similar approaches using absolute qPCR have proven effective in other species for pathogen detection and infection level assessment (4-5), further supporting the value of these tools for developing standardized, cross-species welfare monitoring protocols in aquaculture. Additiona lly, while metagenomics analysis provided insights into microbial communities associated with skin lesions, proteomic profiling of skin mucus allowed exploration of how aquarium maintenance conditions and senescence alter the mucus proteom e. Also, it revealed a wide array of bioactive peptides with potential antimicrobial and immunological roles, which, similar to findings in fish and other molluscs, can serve as reliable indicators of physiological status and stress (6), supporting their potential use as OWIs in octopus. These findings highlight skin mucus as a promising, non-invasive source of biomarkers for health and welfare monitoring in octopus aquaculture.
References
1. Powell AL., 2022. 2. Gestal C. et al. 2007. 3 . Farto et al., 2003. 4. Yang et al., 2014. 5. Truong and Šlapeta , 2023. 6. Almeida et al., 2020.