Impulsive pile driving sound does not induce hearing loss in the longfin squid (Doryteuthis pealeii)a).

Offshore windfarms are a key means to produce clean energy as we seek to limit climate change effects. Impulsive pile driving used for their construction in shallow water environments is among the most intense anthropogenic sound sources. There is an increasing understanding that an array of marine invertebrates detects acoustic cues, yet little is known about how pile driving sound could impact their sound detection abilities. We experimentally quantified potential changes in sound sensitivity for an abundant, commercially and ecologically important squid species (Doryteuthis pealeii) exposed to actual in situ pile driving. The pile was 0.3-m diameter and 10-m long; hammer energy reached 16 kJ per strike. Sound detection thresholds were determined using auditory evoked potentials in animals with no exposure, after one 15-min or five repeated 15-min long pile driving sound sequences, corresponding to cumulative sound exposure levels of 110 and 131 dB re (1 µm s-2)2 s for acceleration and 187 and 214 dB re (1 µPa)2 s for pressure. We found no statistical evidence of temporary threshold shifts in any squid exposed to pile driving sound sequences. These results, combined with companion behavioral studies, suggest that squid may be robust to the sound impacts during offshore windfarm construction.

Stress produces negative judgement bias in cuttlefish.

Judgement bias tasks (JBTs) are used to assess the emotional state and welfare of animals in zoos, farms and laboratories, based on the interpretation of an ambiguous or intermediate cue. Animals in positive affective states are more likely to interpret the ambiguous cue positively, whereas animals experiencing negative affect are more likely to interpret ambiguous cues pessimistically. Here, we developed a modified JBT assay for the stumpy-spined cuttlefish, Sepia bandensis, to determine whether cuttlefish exhibit negative affective states resulting from external stressors. Positive and neutral visual cues were presented twice daily until animals learned to associate food with the reinforced visual cue. After training, one treatment group was exposed to combined exposure and handling stress produced by 6 days of impoverished housing and simulated net capture. Our control group received no stress experience. In test trials performed after the stress experience, stressed animals showed higher latencies to approach ambiguous cues, spent significantly less time in rooms with ambiguous cues once they entered, and were less likely to enter first into the ambiguous cue-paired room compared with controls. These behaviours suggest that stress induces pessimistic judgement bias in cuttlefish, the first indication of this capacity in cephalopods.

The departure of sperm whales (Physeter macrocephalus) in response to the declining jumbo squid (Dosidicus gigas) population in the central portion of the Gulf of California.

As sperm whales are important predators that control energy flux in the oceans, changes in their population can be used as a sentinel to measure of ecosystem health. The present study conducted a sperm whale survey of the eastern Midriff Islands Region in the Gulf of California over the course of nine years, recording sightings and collecting photographs of the fluke of sperm whale individuals. A photo-identification catalog was compiled, while individual recapture data were used to estimate the population size in the central portion of the Gulf of California, using a Jolly-Seber POPAN open population model. The results obtained show a yearly population of between 20 and 167 sperm whales, with a super population of 354 sperm whales observed between 2009 and 2015. However, from 2016 to 2018, no sightings of the species were recorded, which coincides with the decline observed in landings of their main prey, the jumbo squid, in the region. General additive model conducted on sperm whale sightings per unit of effort vs jumbo squid landings obtained an adjusted R2 of 0.644 and a deviance explained of 60.3%, indicating a good non-linear relationship between sightings of this odontocete and its prey availability. This evidence suggests that sperm whales departed the region between 2016 and 2018, due to a documented fishery collapse alongside changes of their main prey into its small phenotype, possibly as the result of increase warming conditions in surface and subsurface waters in the Gulf of California in the last three decades.

Membrane structures and functional correlates in the bi-segmented eye lens of the cephalopod.

The cephalopod eye lens is unique because it has evolved as a compound structure with two physiologically distinct segments. However, the detailed ultrastructure of this lens and precise optical role of each segment are far from clear. To help elucidate structure-function relationships in the cephalopod lens, we conducted multiple structural investigations on squid. Synchrotron x-ray scattering and transmission electron microscopy disclose that an extensive network of structural features that resemble cell membrane complexes form a substantial component of both anterior and posterior lens segments. Optically, the segments are distinct, however, and Talbot interferometry indicates that the posterior segment possesses a noticeably higher refractive index gradient. We propose that the hitherto unrecognised network of membrane structures in the cephalopod lens has evolved to act as an essential conduit for the internal passage of ions and other metabolic agents through what is otherwise a highly dense structure owing to a very high protein concentration.

Developmental gene expression in the eyes of the pygmy squid Xipholeptos notoides.

The eyes of squids, octopuses, and cuttlefish are a textbook example for evolutionary convergence, due to their striking similarity to those of vertebrates. For this reason, studies on cephalopod photoreception and vision are of importance for a broader audience. Previous studies showed that genes such as pax6, or certain opsin-encoding genes, are evolutionarily highly conserved and play similar roles during ontogenesis in remotely related bilaterians. In this study, genes that encode photosensitive proteins and Reflectins are identified and characterized. The expression patterns of rhodopsin, xenopsin, retinochrome, and two reflectin genes have been visualized in developing embryos of the pygmy squid Xipholeptos notoides by in situ hybridization experiments. Rhodopsin is not only expressed in the retina of X. notoides but also in the olfactory organ and the dorsal parolfactory vesicles, the latter a cephalopod apomorphy. Both reflectin genes are expressed in the eyes and in the olfactory organ. These findings corroborate previous studies that found opsin genes in the transcriptomes of the eyes and several extraocular tissues of various cephalopods. Expression of rhodopsin, xenopsin, retinochrome, and the two reflectin genes in the olfactory organ is a finding that has not been described so far. In other organisms, it has been shown that Retinochrome and Rhodopsin proteins are obligatorily associated with each other as both molecules rely on each other for Retinal isomerisation. In addition, we demonstrate that retinochrome is expressed in the retina of X. notoides and in the olfactory organ. This study shows numerous new expression patterns for Opsin-encoding genes in organs that have not been associated with photoreception before, suggesting that either Opsins may not only be involved in photoreception or organs such as the olfactory organ are involved in photoreception.

Cuttlefish ink-derived melanin nanoparticle-embedded tremella fuciformis polysaccharide hydrogels for the treatment of MRSA-infected diabetic wounds.

Diabetic wounds arise great attention as they are difficult to heal and easily suffer from serious bacterial infection. However, the overuse of antibiotics increases the resistance of bacteria and makes common drugs ineffective. Here, we developed a photothermal hydrogel (TFP/NP) composed of tremella fuciformis polysaccharides (TFPs) and cuttlefish ink-derived melanin nanoparticles (NPs). The NPs can produce reliable photothermal effects under near-infrared laser (NIR) irradiation and help to remove the bacteria in the wounds, while TFPs were able to form hydrogel frameworks which possessed anti-inflammatory effects and could be applied to promote wound healing. The TFP/NP hydrogels produced stable thermal effects under NIR irradiation and could continuously kill bacteria. The experiment on a full-layer skin wound sMRSA activity and could improve the healing efficiency. The wounds of the mice could be repaired within 14 days after reasonable treatment. In addition, the hydrogels play significant roles in promoting collagen deposition, anti-inflammation, angiogenesis, and cell proliferation during the therapeutic process. This research provides a simple and effective method for the therapy of bacterial infection wounds through the synergistic effect of TFPs and NPs.

Ontogenetic, Sexual, and Monthly Niche Segregation of Sepia esculenta in the Northern East China Sea Revealed by Stable Carbon and Nitrogen Isotopes.

Golden cuttlefish play a significant role in the food web of the East and Yellow Seas and are a valuable fishery resource in Chinese coastal waters. Samples of golden cuttlefish were obtained from the northern East China Sea between September 2021 and March 2022, and stable isotope methods were utilized in this study to examine the variations in the forage ecology of golden cuttlefish. Our findings reveal dynamic shifts in carbon and nitrogen stable isotopes (δ13C and δ15N), highlighting intricate foraging strategies tailored to growth and environmental changes. A notable trend emerges: an initial growth-linked rise in δ13C and δ15N enrichment, followed by seasonal fluctuations mirroring seasonal food availability. The ontogenetic niche evolution displays striking habitat shifts and trophic level escalation in small mantle length stages, transitioning to niche overlap and subtle trophic shifts later on. Sex-specific differences emerge, with females occupying higher trophic levels than males in most samples. This comprehensive study underscores the complexity and adaptability of golden cuttlefish feeding ecology, inviting further inquiry into their intricate relationships within the marine ecosystem.

A Japanese Case of Food Protein-induced Enterocolitis Syndrome Caused by Multiple Seafoods.

Food protein-induced enterocolitis syndrome (FPIES) caused by fish and others is prevalent in the Mediterranean regions but is less frequently reported in Japan. This case report describes a 3-year-old Japanese girl who developed FPIES triggered by multiple seafoods, including swordfish, cod, and squid. The diagnosis was confirmed through oral food challenge tests (OFC), which led to repeated vomiting and an increase in thymus and activation-regulated chemokine (TARC) levels. This case highlights the importance of considering fish-induced FPIES in the differential diagnosis of recurrent vomiting in children and suggests the potential utility of TARC levels in diagnosing and monitoring FPIES.

Mesoscale eddies exert inverse latitudinal effects on global industrial squid fisheries.

Squid species, as a burgeoning global food source, has garnered significant concerns due to expanding fisheries and little regulation. Elucidating the dynamics of squid fisheries and their biophysical coupling mechanisms is crucial for predicting spatiotemporal variations in squid fisheries and their sustainable management. Mesoscale eddies are discrete rotating oceanographic features that dominate local environmental variations and have been shown to modulate top predators. However, given controls of both predators and environmental factors, it remains unknown how eddies impact mid-trophic level species such as squids. Using satellite-based global squid fishery datasets, we showed an inverse latitudinal pattern of eddy-induced squid fisheries, where fishing activities are aggregated in (repelled from) cyclonic (anticyclonic) eddy cores in tropical waters and anticyclonic (cyclonic) eddy cores in temperate waters, and this pattern can be significantly enhanced with increasing eddy amplitude. Regarding solely the satellite-based global squid fisheries, eddy-induced environmental variations may generate a trade-off between food intake and energy expenditure, causing these oceanic squids to prefer cool cyclonic eddies in hot but food-limited waters, and warm anticyclonic eddies in nutritious but heat-limited waters. Given that eddy activity is projected to continuously enhance under global warming, our finding of eddy-driven bottom-up control for squid fisheries highlights an increasingly important hotspot for squid stock predictions and ecosystem-based ocean management in a changing climate.

Insights on long-term ecosystem changes from stable isotopes in historical squid beaks.

BACKGROUND: Assessing the historical dynamics of key food web components is crucial to understand how climate change impacts the structure of Arctic marine ecosystems. Most retrospective stable isotopic studies to date assessed potential ecosystem shifts in the Arctic using vertebrate top predators and filter-feeding invertebrates as proxies. However, due to long life histories and specific ecologies, ecosystem shifts are not always detectable when using these taxa. Moreover, there are currently no retrospective stable isotopic studies on various other ecological and taxonomic groups of Arctic biota. To test whether climate-driven shifts in marine ecosystems are reflected in the ecology of short-living mesopredators, ontogenetic changes in stable isotope signatures in chitinous hard body structures were analysed in two abundant squids (Gonatus fabricii and Todarodes sagittatus) from the low latitude Arctic and adjacent waters, collected between 1844 and 2023. RESULTS: We detected a temporal increase in diet and habitat-use generalism (= opportunistic choice rather than specialization), trophic position and niche width in G. fabricii from the low latitude Arctic waters. These shifts in trophic ecology matched with the Atlantification of the Arctic ecosystems, which includes increased generalization of food webs and higher primary production, and the influx of boreal species from the North Atlantic as a result of climate change. The Atlantification is especially marked since the late 1990s/early 2000s. The temporal patterns we found in G. fabricii's trophic ecology were largely unreported in previous Arctic retrospective isotopic ecology studies. Accordingly, T. sagittatus that occur nowadays in the high latitude North Atlantic have a more generalist diet than in the XIXth century. CONCLUSIONS: Our results suggest that abundant opportunistic mesopredators with short life cycles (such as squids) are good candidates for retrospective ecology studies in the marine ecosystems, and to identify ecosystem shifts driven by climate change. Enhanced generalization of Arctic food webs is reflected in increased diet generalism and niche width in squids, while increased abundance of boreal piscivorous fishes is reflected in squids' increased trophic position. These findings support opportunism and adaptability in squids, which renders them as potential winners of short-term shifts in Arctic ecosystems.

A synergistic solution for fighting fraudulent practices in squid using light stable isotope ratios and lanthanide tracers.

To identify a novel optimized strategy for preventing fraudulent substitutions of squid species and origins, forty European squids (Loligo vulgaris) and forty flying squids (Todarodes sagittatus) from the Mediterranean Sea and Atlantic Ocean were analyzed for δ13C, δ15N, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Yb, and Lu using isotope ratio mass spectrometry and inductively coupled plasma-mass spectrometry. While δ13C and δ15N variations were mainly species-related, they alone could not reliably distinguish samples. To address this issue, decision rules were developed using Classification and Regression Tree analysis. Threshold values for δ13C (-19.91‰), δ15N (14.87‰), and Pr (0.49 µg kg-1) enabled successful discrimination among Mediterranean European squids, Atlantic European squids, Mediterranean flying squids, and Atlantic flying squids, achieving over 90% accuracy, 81% precision, 80% sensitivity, and 93% specificity. This method holds promise for enhancing traceability and safety in the seafood industry, ensuring product integrity and consumer trust.

Preparation, typical structural characteristics and relieving effects on osteoarthritis of squid cartilage type II collagen peptides.

The promoting effects of collagen and its derivatives on bone health have been uncovered. However, the structure and effects of type II collagen peptides from squid cartilage (SCIIP) on osteoarthritis still need to be clarified. In this study, SCIIP was prepared from squid throat cartilage with pretreatment by 0.2 mol/L NaOH at a liquid-solid ratio of 10:1 for 18 h and hydrolyzation using alkaline protease and flavourzyme at 50 °C for 4 h. The structure of SCIIP was characterized as a molecular weight lower than 5 kDa (accounting for 87.7 %), a high glycine level of 35.0 %, typical FTIR and CD features of collagen peptides, and a repetitive sequence of Gly-X-Y. GP(Hyp)GPD and GPAGP(Hyp)GD were separated and identified from SCIIP, and their binding energies with TLR4/MD-2 were - 8.4 and - 8.0 kcal/mol, respectively. SCIIP effectively inhibited NO production in RAW264.7 macrophages and alleviated osteoarthritis in rats through the TLR4/NF-κB pathway. Therefore, SCIIP exhibited the potential for application as an anti-osteoarthritis supplement.

Lipidomics profile and volatile compounds of squids (Illex argentinus, Ommastrephes Bartram and Dosidicus gigas) in the Argentine, North Pacific Ocean, Equator and Peru by UPLC-triple TOF-MS and HS-SPME-GC-O-MS.

Comprehensive lipid and volatile compound analyses were performed with squids collected from four varied geographical locations to discriminate the regional characteristics. A total of 1442 lipid molecules and 110 volatiles were detected in the squid muscle samples. There were significant differences in the lipid profiles between Argentine squid (Illex argentinus, AGT), North Pacific Ocean squid (Ommastrephes Bartram, NPO), Equatorial squid (Dosidicus gigas, EQ), and Peruvian squid (Dosidicus gigas, PR) muscle. Phosphatidylcholines (14.64%), triacylglycerols (12.42%), and ceramides (10.97%) were the main lipid components. The contents of polyunsaturated fatty acid in phospholipids and in glycerolipids were 30.35-52.05% and 18.11-25.15%, respectively. The volatiles in squids exhibited significant regional variation; 1-pentanol and 1-octanol, 2-ethyl-1-hexanol and terpinen-4-ol, 2,7-ethyl-1-hexanol, 3-methy-1-butanol and 2-propyl-1-pentanol were identified as characteristic flavor compounds in AGT, NPO, EQ, and PR, respectively. Sphingomyelin, phosphatidylserine, phosphatidylethanolamine, and ceramide were strongly correlated with volatiles in squid muscle. Our study is a reference for the lipid nutritional value and flavor compounds of squids.

Peracetic acid treatment of squid eggs infected with parasitic copepod (Ikanecator primus gen. et sp. nov.).

Having been successfully bred in semi-intensive and intensive aquaculture systems, oval squids of the Sepioteuthis lessoniana species complex are emerging as promising candidates for research and industry. Nevertheless, information about pathogens and diseases that may affect squid aquaculture remains sparse. In this study, we identify new parasitic copepod species that causes squid mortality and decreases squid hatching rates, and we also offer a solution to eliminate the pathogen during incubation of squid eggs. The newly discovered copepod Ikanecator primus gen. et sp. nov. was identified on oval squid eggs for the first time using both morphological and molecular diagnostic markers. In the genomes of the copepod and associated microbiome, we identified multiple genes for enzymes involved in cephalopod eggshell degradation in genomes of the copepod and associated microbiome. Furthermore, we conducted experiments to assess efficacy of peracetic acid in inhibiting the I. primus gen. et sp. nov. both in vitro and in vivo using immersion treatment. We established that a 2-min exposure to a concentration of 250 µl/L of peracetic acid containing product (PAA-product; 35 mg/L PAA and 15 mg/L H2O2) inhibited the development of nauplii in vitro. All parasites exposed to a concentration of 500 µl/L of PAA-product (70 mg/L PAA and 30 mg/L H2O2) were eliminated within two minutes. On top of this, the immersion treatment with 500 µl/L of PAA-product (70 mg/L PAA and 30 mg/L H2O2) improved survival of squid embryos and increased size of squid hatchlings compared with control and the immersion treatment with 125 µl/L of PAA-product (17.5 mg/L PAA and 7.5 mg/L H2O2) and the immersion treatment with 250 µl/L of PAA-product (35 mg/L PAA and 15 mg/L H2O2). These findings suggest that PAA holds a great potential as inhibitor and controller of parasitic copepod infections and for overall health management in cephalopod culture.

Transcriptomic responses in the nervous system and correlated behavioural changes of a cephalopod exposed to ocean acidification.

BACKGROUND: The nervous system is central to coordinating behavioural responses to environmental change, likely including ocean acidification (OA). However, a clear understanding of neurobiological responses to OA is lacking, especially for marine invertebrates. RESULTS: We evaluated the transcriptomic response of the central nervous system (CNS) and eyes of the two-toned pygmy squid (Idiosepius pygmaeus) to OA conditions, using a de novo transcriptome assembly created with long read PacBio ISO-sequencing data. We then correlated patterns of gene expression with CO2 treatment levels and OA-affected behaviours in the same individuals. OA induced transcriptomic responses within the nervous system related to various different types of neurotransmission, neuroplasticity, immune function and oxidative stress. These molecular changes may contribute to OA-induced behavioural changes, as suggested by correlations among gene expression profiles, CO2 treatment and OA-affected behaviours. CONCLUSIONS: This study provides the first molecular insights into the neurobiological effects of OA on a cephalopod and correlates molecular changes with whole animal behavioural responses, helping to bridge the gaps in our knowledge between environmental change and animal responses.

Stem-Cell-Regenerative and Protective Effects of Squid (Symplectoteuthis oualaniensis) Skin Collagen Peptides against H2O2-Induced Fibroblast Injury.

Recently, there has been a growing interest in collagen peptides derived from marine sources for their notable ability to protect skin cells against apoptosis induced by oxidants. Therefore, the current study aimed to investigate the fundamental properties of collagen peptides, including their physicochemical, thermal, structural, stem-cell-regenerative, and skin-cell-protective effects, in comparison to commercial collagen peptides. The acid-soluble (ASC) and pepsin-soluble (PSC) collagens exhibited three distinct bands on SDS-PAGE, namely α (α1 and α2), ß, and γ chains, confirming a type I pattern. The thermal profiles obtained from TG and DSC analyses confirmed the denaturation of PSC and ASC at temperatures ranging from 51.94 to 56.4 °C and from 52.07 to 56.53 °C, respectively. The purified collagen peptides were analyzed using SDS-PAGE and MALDI-TOF mass spectrometry, revealing a mass range of 900-15,000 Da. Furthermore, the de novo peptide sequence analysis confirmed the presence of the Gly-X-Y repeating sequence in collagen peptides. Collagen peptide treatments significantly enhanced HFF-1 cell proliferation and migration compared to the control group. ELISA results confirmed the potential interactions between collagen peptides and HFF-1 cells through α2ß1, α10ß1, and α11ß1 integrin receptors. Notably, collagen peptide treatment effectively restored the proliferation of HFF-1 cells damaged by H2O2. Consequently, the advantageous characteristics of squid skin collagen peptides highlight their promising role in regenerative medicine.

State-dependent dynamics of cuttlefish mantle activity.

Cuttlefish skin is a powerful rendering device, capable of producing extraordinary changes in visual appearance over a broad range of temporal scales. This unique ability is typically associated with camouflage; however, cuttlefish often produce skin patterns that do not appear connected with the surrounding environment, such as fast large-scale fluctuations with wave-like characteristics. Little is known about the functional significance of these dynamic patterns. In this study, we developed novel tools for analyzing pattern dynamics, and demonstrate their utility for detecting changes in feeding state that occur without concomitant changes in sensory stimulation. Under these conditions, we found that the dynamic properties of specific pattern components differ for different feeding states, despite no measurable change in the overall expression of those components. Therefore, these dynamic changes are not detectable by conventional analyses focusing on pattern expression, requiring analytical tools specifically targeted to pattern dynamics.

Immobilized chitinase as effective biocatalytic platform for producing bioactive di-N-acetyl chitobiose from recycled chitin food waste.

Described is chitinase immobilization on magnetic nanoparticles (MNPs) as biocompatible support for enzymatic production of di-N-acetyl chitobiose from chitin waste. Chitinase immobilization was feasible with an immobilization yield of 88.9 ± 1.6 % with 97.8 ± 1.0 % retention of activity and compared to free enzyme work, immobilization conferred better thermal and storage stability. As practical benefit the attachment to magnetic nanocarriers enabled easy enzyme recovery after repeated application runs and thus sustainable reuse. In fixed state chitinase retained a remarkable 39.7 ± 2.6 % of the starting activity after 16 reaction cycles. Furthermore, immobilized chitinase showed higher catalytic activity than free chitinase in converting shrimp shells and squid-pens chitins into di-N-acetyl chitobiose in a single-step reaction. The final yield of purified compound was 37.0 ± 1.2 % from shrimp shells and 61.1 ± 0.5 % from squid-pens chitin. In conclusion, an efficient MNP-based chitinase immobilization system with the potential for large-scale production was developed.

Corrected and republished from: "Vibrio fischeri Possesses Xds and Dns Nucleases That Differentially Influence Phosphate Scavenging, Aggregation, Competence, and Symbiotic Colonization of Squid".

Cells of Vibrio fischeri colonize the light organ of Euprymna scolopes, providing the squid bioluminescence in exchange for nutrients and protection. The bacteria encounter DNA-rich mucus throughout their transition to a symbiotic lifestyle, leading us to hypothesize a role for nuclease activity in the colonization process. In support of this, we detected abundant extracellular nuclease activity in growing cells of V. fischeri. To discover the gene(s) responsible for this activity, we screened a V. fischeri transposon mutant library for nuclease-deficient strains. Interestingly, only one strain, whose transposon insertion mapped to nuclease gene VF_1451, showed a complete loss of nuclease activity in our screens. A database search revealed that VF_1451 is homologous to the nuclease-encoding gene xds in Vibrio cholerae. However, V. fischeri strains lacking xds eventually revealed slight nuclease activity on plates upon prolonged incubation. This led us to hypothesize that a second secreted nuclease, identified through a database search as VF_0437, a homolog of V. cholerae dns, might be responsible for the residual nuclease activity. Here, we show that Xds and/or Dns are involved in essential aspects of V. fischeri biology, including natural transformation, aggregation, and phosphate scavenging. Furthermore, strains lacking either nuclease were outcompeted by the wild type for squid colonization. Understanding the specific role of nuclease activity in the squid colonization process represents an intriguing area of future research.IMPORTANCEFrom soil and water to host-associated secretions such as mucus, environments that bacteria inhabit are awash in DNA. Extracellular DNA (eDNA) is a nutritious resource that microbes dedicate significant energy to exploit. Calcium binds eDNA to promote cell-cell aggregation and horizontal gene transfer. eDNA hydrolysis impacts the construction of and dispersal from biofilms. Strategies in which pathogens use nucleases to avoid phagocytosis or disseminate by degrading host secretions are well-documented; significantly less is known about nucleases in mutualistic associations. This study describes the role of nucleases in the mutualism between Vibrio fischeri and its squid host Euprymna scolopes. We find that nuclease activity is an important determinant of colonization in V. fischeri, broadening our understanding of how microbes establish and maintain beneficial associations.

Isolation, characterization, and application of bacteriophage on Vibrio parahaemolyticus biofilm to control seafood contamination.

OBJECTIVE: This study intended to isolate a Vibrio-particular phage from the natural environment, analyse its characteristics and genome sequence, and investigate its reduction effect on V. parahaemolyticus biofilm as a biocontrol agent in squid and mackerel. METHODS: Among 21 phages, phage CAU_VPP01, isolated from beach mud, was chosen for further experiments based on host range and EOP tests. When examining the reduction effect of phage CAU_VPP01 against Vibrio parahaemolyticus biofilms on surfaces (stainless steel [SS] and polyethylene terephthalate [PET]) and food surfaces (squid and mackerel). RESULTS: The phage showed the most excellent reduction effect at a multiplicity-of-infection (MOI) 10. Three-dimensional images acquired with confocal laser scanning microscopy (CLSM) analysis were quantified using COMSTAT, which showed that biomass, average thickness, and roughness coefficient decreased when treated with the phage. Colour and texture analysis confirmed that the quality of squid and mackerel was maintained after the phage treatment. Finally, a comparison of gene expression levels determined by qRT-PCR analysis showed that the phage treatment induced a decrease in the gene expression of flaA, vp0962, andluxS, as examples. CONCLUSION: This study indicated that Vibrio-specific phage CAU_VPP01 effectively controlled V. parahaemolyticus biofilms under various conditions and confirmed that the isolated phage could possibly be used as an effective biocontrol weapon in the seafood manufacturing industry.