Better late than never: Optimising the proteomic analysis of field-collected octopus.

Proteomics, the temporal study of proteins expressed by an organism, is a powerful technique that can reveal how organisms respond to biological perturbations, such as disease and environmental stress. Yet, the use of proteomics for addressing ecological questions has been limited, partly due to inadequate protocols for the sampling and preparation of animal tissues from the field. Although RNAlater is an ideal alternative to freezing for tissue preservation in transcriptomics studies, its suitability for the field could be more broadly examined. Moreover, existing protocols require samples to be preserved immediately to maintain protein integrity, yet the effects of delays in preservation on proteomic analyses have not been thoroughly tested. Hence, we optimised a proteomic workflow for wild-caught samples. First, we conducted a preliminary in-lab test using SDS-PAGE analysis on aquaria-reared Octopus berrima confirming that RNAlater can effectively preserve proteins up to 6 h after incubation, supporting its use in the field. Subsequently, we collected arm tips from wild-caught Octopus berrima and preserved them in homemade RNAlater immediately, 3 h, and 6 h after euthanasia. Processed tissue samples were analysed by liquid chromatography tandem mass spectrometry to ascertain protein differences between time delay in tissue preservation, as well as the influence of sex, tissue type, and tissue homogenisation methods. Over 3500 proteins were identified from all tissues, with bioinformatic analysis revealing protein abundances were largely consistent regardless of sample treatment. However, nearly 10% additional proteins were detected from tissues homogenised with metal beads compared to liquid nitrogen methods, indicating the beads were more efficient at extracting proteins. Our optimised workflow demonstrates that sampling non-model organisms from remote field sites is achievable and can facilitate extensive proteomic coverage without compromising protein integrity.

Tracking the provenance of octopus using isotopic and multi-elemental analysis.

Octopus play an increasingly important role in ocean ecosystems and global fisheries, yet techniques for authenticating provenance are sorely lacking. For the first time, we investigate whether chemical profiling can distinguish geographical origins of octopus on international and domestic scales. Our samples consisted of wild-caught octopus from south-east Asia and southern Australia, regions with high seafood trade. We used a novel combination of stable carbon (δ13C) and oxygen (δ18O) isotope analyses (Isotope-Ratio Mass Spectrometry) of internal calcified structures called statoliths, with elemental analyses (X-Ray Fluorescence using Itrax) of soft-tissue. We found that multivariate profiles exhibited distinctive regional signatures, even across species, with high classification success (∼95%) back to region of origin. This study validates isotopic and multi-elemental profiling as an effective provenance tool for octopus, which could be used to support transparency and accountability of seafood supply chains and thus encourage sustainable use of ocean resources.

Experimental support towards a metabolic proxy in fish using otolith carbon isotopes.

Metabolic rate underpins our understanding of how species survive, reproduce and interact with their environment, but can be difficult to measure in wild fish. Stable carbon isotopes (δ13C) in ear stones (otoliths) of fish may reflect lifetime metabolic signatures but experimental validation is required to advance our understanding of the relationship. To this end, we reared juvenile Australasian snapper (Chrysophrys auratus), an iconic fishery species, at different temperatures and used intermittent-flow respirometry to calculate standard metabolic rate (SMR), maximum metabolic rate (MMR) and absolute aerobic scope (AAS). Subsequently, we analysed δ13C and oxygen isotopes (δ18O) in otoliths using isotope-ratio mass spectrometry. We found that under increasing temperatures, δ13C and δ18O significantly decreased, while SMR and MMR significantly increased. Negative logarithmic relationships were found between δ13C in otoliths and both SMR and MMR, while exponential decay curves were observed between proportions of metabolically sourced carbon in otoliths (Moto) and both measured and theoretical SMR. We show that basal energy for subsistence living and activity metabolism, both core components of field metabolic rates, contribute towards incorporation of δ13C into otoliths and support the use of δ13C as a metabolic proxy in field settings. The functional shapes of the logarithmic and exponential decay curves indicated that physiological thresholds regulate relationships between δ13C and metabolic rates due to upper thresholds of Moto Here, we present quantitative experimental evidence to support the development of an otolith-based metabolic proxy, which could be a powerful tool in reconstructing lifetime biological trends in wild fish.