Cleaner Fish Research Articles

The effect of serum-free media on the metabolic yields and growth rates of C2C12 cells in the context of cultivated meat production

Successful commercialization of cultivated meat products as alternative sources of dietary protein for a growing global population will require development of efficient and inexpensive serum-free cell culture media. It is known that serum-free media elicit dissimilar cell growth behavior compared to serum-based media, but data comprehensively exploring the effects of serum-free culture on the nutrient requirements of cultivated meat-relevant cell types have yet to be reported. We performed spent media analysis of C2C12 cells growing in Essential 8 serum-free media and in conventional serum-containing media. Data indicated that although the cell growth rates were similar in the two media over seven days, there were significant differences in the utilization rates of some key nutrients such as glucose, glutamine, glycine, and cystine. We extrapolated from the data that roughly 250–275 g of amino acids and 1100–1500 g of glucose would be required to produce 1 kg of C2C12 cells, though these requirements may change when using more optimized cells, media, and feed strategies. Our results highlight the influence of serum and serum replacements on cell metabolism, and indicate that previous data from metabolic studies performed using serum-containing media may not directly translate to serum-free systems.

How to establish detection limits for environmental microplastics analysis

Establishing analytical detection limits is crucial. Common methods to do so are suitable only for variables with continuous distributions. Because count data for microplastic particles is a discrete variable following the Poisson distribution, currently-used approaches for estimating the detection limit in microplastics analysis are inadequate. Here we evaluate detection limits with techniques for low-level discrete observations to develop proper approaches for estimating the minimum detectable amount (MDA) in microplastic particle analysis, using blank sample data from an interlaboratory calibration exercise for clean water (representing drinking water), dirty water (ambient water), sediment (porous media) and fish tissue (biotic tissues). Two MDAs are applicable: MDAA to evaluate analytical methods, estimated with replicate blank data; and MDAB for individual sample batches, calculated with a single blank count. For illustrative purposes, this dataset's overall MDAA values were 164 counts (clean water), 88 (dirty water), 192 (sediment), and 379 (tissue). MDA values should be reported on a laboratory-specific basis and for individual size fractions, as this provides more useful information about capabilities of individual laboratories. This is due to wide variation in blank levels, as noted by MDAB values (i.e., among different laboratories) from 14 to 158 (clean water), 9 to 86 (dirty water, 9 to 186 (sediment), and 9 to 247 (tissue). MDA values for fibers were considerably greater than for non-fibers, suggesting that separate MDA values should be reported. This study provides a guideline for estimation and application of microplastics MDA for more robust data to support research activities and environmental management decisions.

Tissue Engineering Challenges for Cultivated Meat to Meet the Real Demand of a Global Market.

Cultivated meat (CM) technology has the potential to disrupt the food industry-indeed, it is already an inevitable reality. This new technology is an alternative to solve the environmental, health and ethical issues associated with the demand for meat products. The global market longs for biotechnological improvements for the CM production chain. CM, also known as cultured, cell-based, lab-grown, in vitro or clean meat, is obtained through cellular agriculture, which is based on applying tissue engineering principles. In practice, it is first necessary to choose the best cell source and type, and then to furnish the necessary nutrients, growth factors and signalling molecules via cultivation media. This procedure occurs in a controlled environment that provides the surfaces necessary for anchor-dependent cells and offers microcarriers and scaffolds that favour the three-dimensional (3D) organisation of multiple cell types. In this review, we discuss relevant information to CM production, including the cultivation process, cell sources, medium requirements, the main obstacles to CM production (consumer acceptance, scalability, safety and reproducibility), the technological aspects of 3D models (biomaterials, microcarriers and scaffolds) and assembly methods (cell layering, spinning and 3D bioprinting). We also provide an outlook on the global CM market. Our review brings a broad overview of the CM field, providing an update for everyone interested in the topic, which is especially important because CM is a multidisciplinary technology.

Genetic study reveals local differentiation persisting in the face of high connectivity and a genomic inversion likely linked with sexual antagonism in a common marine fish

Abstract Sustainable harvest of wild populations requires knowledge of the underlying population structure. The focus of this study is on goldsinny wrasse (Ctenolabrus rupestris), a small marine fish inhabiting coastal waters of the north-eastern Atlantic. This species is caught in large numbers to serve as cleaner fish in salmonid aquaculture. We genotyped 2073 goldsinny wrasse from 43 sites along the Scandinavian coastline with 143 SNPs. Seven of the SNPs were linked and likely reside within a large genomic inversion dominated by one haplotype. The heterokaryotype of the putative inversion displayed sex-specific growth patterns, potentially resolving sexual antagonism for this trait. The unlinked 134 SNPs showed modest isolation-by-distance with samples from the northernmost locations showing highest divergence, whereas sites farther south were much more interconnected. Genetic divergence (FST) was highly variable among sites within regions, suggesting a varying degree of connectivity and local divergence. We conclude that despite a high degree of gene-flow mediated through pelagic dispersal in early life stages, regional and some local population structure remains due to limited adult movement in addition to other unidentified factors. Consequently, the species might be more vulnerable to local disturbances than previously anticipated.

Pumpkin seed proteins rival animal gelatin in increasing the cytoaffinity of edible microbeads for cell-based meat culture

Edible microbeads are hotly sought after for emerging cell-based meat culture but there are no major breakthroughs so far. Herein we report a functional edible microbead with alginate as core coated with pumpkin proteins as shell. Proteins from 11 plant-seeds were extracted and tested their cytoaffinity as gelatin replacer by grafting them on alginate microbeads and pumpkin seed protein coated microbeads shown the best performance in stimulating proliferation of C2C12 cells (by 17 folds in a week), 3T3-L1 adipocytes, chicken muscle satellite cells and primary porcine myoblast. The cytoaffinity of pumpkin seed protein coated microbeads comparable with that of animal gelatin microbeads. Protein sequencing analysis on pumpkin seed proteins found that it is rich in RGD tripeptide moiety, which are known to be enhance cytoaffinity. Our work advances our search for edible microbeads as ECM materials for cell-based meat culture.

Global, regional, and cryptic population structure in a high gene-flow transatlantic fish.

Lumpfish (Cyclopterus lumpus) is a transatlantic marine fish displaying large population sizes and a high potential for dispersal and gene-flow. These features are expected to result in weak population structure. Here, we investigated population genetic structure of lumpfish throughout its natural distribution in the North Atlantic using two approaches: I) 4,393 genome wide SNPs and 95 individuals from 10 locations, and II) 139 discriminatory SNPs and 1,669 individuals from 40 locations. Both approaches identified extensive population genetic structuring with a major split between the East and West Atlantic and a distinct Baltic Sea population, as well as further differentiation of lumpfish from the English Channel, Iceland, and Greenland. The discriminatory loci displayed ~2-5 times higher divergence than the genome wide approach, revealing further evidence of local population substructures. Lumpfish from Isfjorden in Svalbard were highly distinct but resembled most fish from Greenland. The Kattegat area in the Baltic transition zone, formed a previously undescribed distinct genetic group. Also, further subdivision was detected within North America, Iceland, West Greenland, Barents Sea, and Norway. Although lumpfish have considerable potential for dispersal and gene-flow, the observed high levels of population structuring throughout the Atlantic suggests that this species may have a natal homing behavior and local populations with adaptive differences. This fine-scale population structure calls for consideration when defining management units for exploitation of lumpfish stocks and in decisions related to sourcing and moving lumpfish for cleaner fish use in salmonid aquaculture.

A marine cleaning mutualism provides new insights in biological market dynamics.

Most mutually beneficial social interactions (cooperation within species, mutualism between species) involve some degree of partner choice. In an analogy to economic theory as applied to human trading practices, biological market theory (BMT) focuses on how partner choice affects payoff distributions among non-human traders. BMT has inspired a great diversity of research, including research on the mutualism between cleaner fish Labroides dimidiatus and other marine fish, their 'clients'. In this mutualism, clients have ectoparasites removed and cleaners obtain food in return. We use the available data on L. dimidiatus cleaner-client interactions to identify avenues for future expansion of BMT. We focus on three main topics, namely how partner quality interacts with supply-to-demand ratios to affect service quality, the role of threats and forms of forceful intervention, and the potential role of cognition. We consider it essential to identify the specifics of each biological market as a basis for the development of more sophisticated BMT models. This article is part of the theme issue 'Half a century of evolutionary games: a synthesis of theory, application and future directions'.

Do Cleaner Fish (Labroides dimidiatus) Have General Cognitive Ability? A Reanalysis of Individual Differences Data and Consideration of Phylogenetic Context

Do Cleaner Fish (Labroides dimidiatus) Have General Cognitive Ability? A Reanalysis of Individual Differences Data and Consideration of Phylogenetic Context

Review on cultured meat: ethical alternative to animal industrial farming

It is estimated that around 8% of the entire world population identifies themselves as vegetarians. That leaves us with statistics of more than 90% of the world's population prefers a meat-based diet. Now with the alarmingly increasing population of over 7.8 billion, the demand for meat is skyrocketing. Due to the high demand, factors like land, water and greenhouse emission are becoming unsustainable. Even after taking all these concerns into account, why is it so difficult for us to eliminate/reduce meat from our diet? How is the future generation going to satisfy their meat cravings? However, through recent research, it seems there is an alternative, a solution to all these concerns, cultured/ lab-grown meat. Cultured meat is meat produced by in vitro cell culture of animal cells, instead of from slaughtered animals using various tissue engineering techniques. This invitro meat technology is still at its elementary stages but shows great potential in curbing the problem of livestock overcrowding and its impact on the environment. Cultured meat seems to promise a sustainable and reliable alternative for its consumers. This review aimed to provide an elaborate comprehension of the entire process of the production, processing and commercialization of cultured meat, and also provides an insight on the possible obstacles it encounters, especially overcoming ethical conundrums and social prejudice.

Bacteria of Zoonotic Interest Identified on Edible Freshwater Fish Imported to Australia

Previous research has shown that freshwater edible fish imported into Australia are not compliant with Australian importation guidelines and as a result may be high risk for bacterial contamination. In the present study, the outer surface of imported freshwater fish were swabbed, cultured, confirmatory tests performed and antimicrobial patterns investigated. Channidae fish (Sp. A/n = 66) were contaminated with zoonotic Salmonella sp./Staphylococcus aureus (n = 1/66) and other bacteria implicated in cases of opportunistic human infection, these being Pseudomonas sp. (including P. mendocina and P. pseudoalcaligenes (n = 34/66)); Micrococcus sp. (n = 32/66); Comamonas testosteroni (n = 27/66) and Rhizobium radiobacter (n = 3/66). Pangasiidae fish (Species B/n = 47) were contaminated with zoonotic Vibrio fluvialis (n = 10/47); Salmonella sp. (n = 6/47) and environmental bacteria Micrococcus sp. (n = 3/47). One sample was resistant to all antimicrobials tested and is considered to be Methicillin Resistant S. aureus. Mud, natural diet, or vegetation identified in Sp. A fish/or packaging were significantly associated with the presence of Pseudomonas spp. The study also showed that visibly clean fish (Sp. B) may harbour zoonotic bacteria and that certain types of bacteria are common to fish groups, preparations, and contaminants. Further investigations are required to support the development of appropriate food safety recommendations in Australia.

Soy Protein Amyloid Fibril Scaffold for Cultivated Meat Application.

It is important to have sustainable and edible scaffolds to produce cultivated meat. In this research, three-dimensional (3D) porous scaffolds were developed by soy protein amyloid fibrils for cultivated meat applications. Food-safe biological and physical cross-linking methods using microbial transglutaminase and temperature-controlled water vapor annealing technique were employed to crosslink soy protein amyloid fibrils, resulting in the production of 3D scaffolds. The generated 3D scaffolds had pores with sizes ranging from 50 to 250 μm, porosities of 72-83%, and compressive moduli of 3.8-4.2 kPa, depending on the type of soy protein used in the process (β-conglycinin (7S), glycinin (11S) and soy protein isolate (SPI)). When present with pepsin, these scaffolds can degrade within an hour but remain stable in phosphate-buffered saline for at least 30 days. The soy protein amyloid fibril scaffolds enabled C2C12 mouse skeletal myoblasts proliferate and differentiate without adding cell adhesive proteins or other coatings. The results demonstrate the potential of abundant and inexpensive soy protein amyloid fibrils to be utilized as scaffold materials for cultivated meat in the food industry.

Protein engineering

Protein engineering is the creation of new amino acid sequences to modify proteins. There are many potential applications where the addition, deletion or modification of amino acids can aid or hinder enzyme efficacy. As our knowledge of genetics has expanded rapidly, so has our knowledge of how this might be applied in reality. Proteins are coded for by the 20 amino acids found in nature – should we limit our efforts to those 20 amino acids? This issue also looks at research which is trying to see if we can move beyond this 20-code language of proteins, could engineering offer us new amino acids and new protein functions?Why do we need to do this? The world is under pressure to feed more people in more sustainable ways and treat people with medicine created more efficiently and sustainably. Protein engineering methods might help us reach this goal, producing more sustainable food possibly from unlikely sources, such as from pathogenic bacteria like Yersinia pestis, the causative agent of the plague which could help create lab-grown meat. Sustainable farming could be supported by protein engineering, reducing the carbon footprint of crops and supporting greater yields. I found it interesting that the human race has removed genetic diversity in our crops over time through farming techniques, so we now need to explore new ways of introducing diversity in order to make our crops work harder and be more resilient to pests and climate change.There are many questions and challenges in this area and the articles explore just some of these. They also give you an insight into how this can be applied in reality.As always, this issue also covers personal experiences in biochemistry for anyone interested in careers in the area check out the interviews, which includes interviews with two of the past 10 years' Colworth Medal winners, Professor Helen Walden and Dr Madan Babu, in celebration of the award's 60th anniversary, and write-ups of visits. I always love hearing about what people in the field do daily, as the roles and experiences people have in biochemistry can be so varied.

The skin mucosal barrier of lumpfish (Cyclopterus lumpus L.) is weakened by exposure to potential aquaculture production-related stressors.

Various cleaner fish species, such as the lumpfish (Cyclopterus lumpus L.), are used in the sea cage production of Atlantic salmon (Salmo salar L.) as a control measure against the ectoparasitic salmon louse (Lepeophtheirus salmonis). Nonetheless, during severe lice infestation, alternative treatments are required to control parasitic burden. The aim of this study was to gain insight into how lumpfish skin responds to different chemicals used to treat parasites. The authors collected skin from lumpfish from both research facilities (tank-reared fish) and commercial production (cage-reared fish) and used operational welfare indicators, in vitro models, histology and transcriptomics to study how the skin responded to two anti-parasitic oxidative chemicals, hydrogen peroxide (H2 O2 ) and peracetic acid. Lumpfish sampled from the farm were classified as clinically healthy or weak based on their morbidity status, and fish from each category were used to gain insight into how the therapeutics affect the skin barrier. Differences between healthy and weakened (moribund) fish, and between treated fish from each of the two groups, were observed. Histological examination showed an overall reduced skin quality in fish characterized as moribund, including different grades of exposed bony plates. In vitro oxidant-treated lumpfish skin had reduced the migration capacity of keratocytes, a weakened epidermal barrier, and altered gene transcription, changes that are known predisposing factors to secondary infections. Skin from non-treated, healthy fish sampled from commercial farms exhibited similar features and attributes to oxidant-exposed tank-reared fish from a research facility, suggesting that apparently healthy cage-held lumpfish exhibited stress responses in the epidermal barrier. The results of the study outline the risks and consequences lumpfish can face if accidentally subjected to potential anti-parasitic oxidant treatments aimed at Atlantic salmon. It also strengthens the evidence behind the requirement that lumpfish should be removed from the cages before being potentially exposed to this type of treatment and outlines the potential risks of differing husbandry practices upon lumpfish health, welfare and resilience.

Ginsenoside Rg2 Promotes the Proliferation and Stemness Maintenance of Porcine Mesenchymal Stem Cells through Autophagy Induction

Mesenchymal stem cells (MSCs) can be used as a cell source for cultivated meat production due to their adipose differentiation potential, but MSCs lose their stemness and undergo replicative senescence during expansion in vitro. Autophagy is an important mechanism for senescent cells to remove toxic substances. However, the role of autophagy in the replicative senescence of MSCs is controversial. Here, we evaluated the changes in autophagy in porcine MSCs (pMSCs) during long-term culture in vitro and identified a natural phytochemical, ginsenoside Rg2, that could stimulate pMSC proliferation. First, some typical senescence characteristics were observed in aged pMSCs, including decreased EdU-positive cells, increased senescence-associated beta-galactosidase activity, declined stemness-associated marker OCT4 expression, and enhanced P53 expression. Importantly, autophagic flux was impaired in aged pMSCs, suggesting deficient substrate clearance in aged pMSCs. Rg2 was found to promote the proliferation of pMSCs using MTT assay and EdU staining. In addition, Rg2 inhibited D-galactose-induced senescence and oxidative stress in pMSCs. Rg2 increased autophagic activity via the AMPK signaling pathway. Furthermore, long-term culture with Rg2 promoted the proliferation, inhibited the replicative senescence, and maintained the stemness of pMSCs. These results provide a potential strategy for porcine MSC expansion in vitro.

Ensuring sustainability in sensory sciences

Ensuring sustainability in sensory sciences

Higher well-being individuals are more receptive to cultivated meat: An investigation of their reasoning for consuming cultivated meat

It is evident that over-consumption of meat can contribute to the emission of hazardous greenhouse gases. One viable way to address such climate impact is to make people become more aware of more sustainable diet options, such as cultivated meat. However, it is challenging to instigate change in people's meat-eating habit, and empirical works have been examining the psychological factors that are related to consumers' willingness to consume cultivated meat. Research has suggested that psychological well-being can play a role in the meaning-making of food consumption, with higher well-being individuals showing more recognition of other sociocultural benefits of consuming food beyond just fulfilling their sustenance needs. As existing works have yet to understand the link between well-being and consumption of novel foods, the current research set out to fill this gap by examining the relationship between people's psychological well-being and their willingness to consume cultivated meat via different reasons (mediators) for consuming cultivated meat. We recruited a representative sample of 948 adults in Singapore to complete an online survey. The study offered the first evidence that there is a positive relationship between people's psychological well-being and their willingness to consume cultivated meat. Further, results revealed that their higher willingness can be motivated by the perception that cultivated meat is as healthy and nutritious, as safe as, and has the same sensory quality as real meat, and is beneficial to the society. This investigation adds to the growing literature on consumer acceptance of cultivated meat by showing the novel finding that well-being and receptivity to cultivated meat is positively linked, and such a positive link can be explained by people's better recognition of the prospective benefits offered by this alternative food.

Bioactive cellulose acetate nanofiber loaded with annatto support skeletal muscle cell attachment and proliferation.

Electrospinning emerged as a promising technique to produce scaffolds for cultivated meat in function of its simplicity, versatility, cost-effectiveness, and scalability. Cellulose acetate (CA) is a biocompatible and low-cost material that support cell adhesion and proliferation. Here we investigated CA nanofibers, associated or not with a bioactive annatto extract (CA@A), a food-dye, as potential scaffolds for cultivated meat and muscle tissue engineering. The obtained CA nanofibers were evaluated concerning its physicochemical, morphological, mechanical and biological traits. UV-vis spectroscopy and contact angle measurements confirmed the annatto extract incorporation into the CA nanofibers and the surface wettability of both scaffolds, respectively. SEM images revealed that the scaffolds are porous, containing fibers with no specific alignment. Compared with the pure CA nanofibers, CA@A nanofibers showed increased fiber diameter (420 ± 212nm vs. 284 ± 130nm). Mechanical properties revealed that the annatto extract induces a reduction of the stiffness of the scaffold. Molecular analyses revealed that while CA scaffold favored C2C12 myoblast differentiation, the annatto-loaded CA scaffold favored a proliferative state of these cells. These results suggest that the combination of cellulose acetate fibers loaded with annatto extract may be an interesting economical alternative for support long-term muscle cells culture with potential application as scaffold for cultivated meat and muscle tissue engineering.

Perceptions of Cultivated Meat in Millennial and Generation X Consumers Resident in Aotearoa New Zealand

Evidence suggests that consumer perceptions and acceptance of cultivated meat (CM) differ between countries, cultures, and consumer groups. Limited research specific to Aotearoa New Zealand (A-NZ) is available. Survey responses from 592 A-NZ residents were analysed to determine CM awareness, willingness to engage with CM, and perceived CM product attributes relative to conventional meat and plant-based meat alternatives (PBMAs). The effects of gender, age, meat/PBMA consumption frequency, CM awareness, and food neophobia on CM perceptions were determined. The statements were rated on a seven-point Likert scale. Half (52%) of the respondents agreed they were aware of CM. The awareness was higher in men compared to women (p = 0.036), higher in Millennials compared to Generation X (p = 0.022), and higher in regular compared to infrequent PBMA consumers (p = 0.0003). The willingness to engage with CM and perceived CM product attributes were consistently more positive in consumers who were aware, compared with consumers not aware of CM (p < 0.05). Being male, Millennial, low neophobic and a low meat, or high PBMA consumer was also associated with higher potential engagement and perceptions of CM to varying extents. Segmentation divided the respondents into three groups. The ‘positive’ cluster (41%) consumed more PBMAs and less meat and was more aware of CM than the ‘neutral’ (50%) and ‘negative’ (9%) clusters. In conclusion, consumers in A-NZ are not a homogenous group with regards to their perceptions and potential engagement with CM. Increasing awareness and familiarity with CM will be an important strategy to increase engagement with CM.

The presence of territorial damselfish predicts choosy client species richness at cleaning stations.

Mutualisms are driven by partners deciding to interact with one another to gain specific services or rewards. As predicted by biological market theory, partners should be selected based on the likelihood, quality, reward level, and or services each partner can offer. Third-party species that are not directly involved in the interaction, however, may indirectly affect the occurrence and or quality of the services provided, thereby affecting which partners are selected or avoided. We investigated how different clients of the sharknose goby (Elacatinus evelynae) cleaner fish were distributed across cleaning stations, and asked what characteristics, relating to biological market theory, affected this distribution. Through quantifying the visitation and cleaning patterns of client fish that can choose which cleaning station(s) to visit, we found that the relative species richness of visiting clients at stations was negatively associated with the presence of disruptive territorial damselfish at the station. Our study highlights, therefore, the need to consider the indirect effects of third-party species and their interactions (e.g., agonistic interactions) when attempting to understand mutualistic interactions between species. Moreover, we highlight how cooperative interactions may be indirectly governed by external partners.

Cultured meat, clean meat,… queer meat? A vegan queer ecofeminist perspective on the implications of cellular agriculture

This perspective article briefly explores the social implications of cellular agriculture from a Vegan Queer Ecofeminist point of view by referring to a synthesis of currently agreed-upon (possible) positive and negative effects of post-animal agriculture and highlighting how these effects actually ignore key ethical problems inherent in animal agriculture itself. By invisibilizing these, discussions of cellular-ag remain in danger of obscuring the ways in which an intact and unexamined paradigm based on capitalist, patriarchal speciesism will continue to foment exploitative and unjust practices in a dying planet. The article emphasizes the urgent need to address the complexities of cellular-ag from a multidisciplinary perspective that actively engages with the demands of true global justice for all, nonhuman and human.