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One major difficulty in identifying the gelatinous bodied bullet-shaped Siphonophore, Diphyids, is that their shape is deformed following ethanol fixation. Ethanol often is preferred over other fixatives, since samples fixed in ethanol can be used for molecular studies that can supplement morphological findings. To overcome this problem, we obtained protein mass spectra of ten species of Diphyidae found in the waters of the Kuroshio Current (Northwest Pacific and South Coast of South Korea) to test whether MALDI-ToF MS could be used as a methodology for species identification. In addition, a number of morphological characteristics that can be used with ethanol-treated samples was summarized. Concatenated phylogenetic analysis was also performed to determine the phylogenetic relationship by obtaining partial sequences of four genes (mtCOI, 16S rRNA, 18S rRNA, and ITS regions). Based on our integrative analysis, MALDI-ToF MS was evaluated as a potentially fast, inexpensive, and accurate tool for species identification along with conventional morphological and DNA barcoding for Diphyidae.

Taxonomical research on siphonophores is lacking despite their high biodiversity. We collected siphonophores in Jeju and the southern coasts of Korea and conducted morphological redescriptions using multi-focus stacked digital images. As a result, we revealed eight unrecorded siphonophores (Abyla haeckeli, Ceratocymba leuckartii, Bassia bassensis, Dimophyes arctica, Lensia subtilis, Lensia subtiloides, Muggiaea atlantica, and Nanomia bijuga). Six genera, excluding Muggiaea, were reported for the first time in Korean waters, and subfamily Abylinae, to which Abyla haeckeli and Ceratocymba leuckartii belong, was also first recorded in Korea. The distributions and habitats of these species were summarized, with most of the species having cosmopolitan distributions but variations in individual size depending on environmental conditions. Phylogenetic analysis based on mtCOI was conducted for each family. Finally, we updated the species list of siphonophores in Korean waters to include 3 suborders, 5 families, 3 subfamilies, 14 genera, and 21 species. Our findings constitute baseline data for further research on Korean siphonophores.

Jellyfish exhibit an extraordinary survival strategy in high-density populations that exceed the carrying capacity of ecosystems. It is particularly questionable how jellyfish can thrive in habitats where the concentration of toxic nitrogenous waste (i.e., NH 3 ) in the ambient water could be extremely high caused by high-density populations. However, physiological mechanisms of the survival strategy in jellyfish remain poorly understood, especially at the polyp stage, even though this stage is a key role in jellyfish population dynamics. In the present study, we investigated the trophic isotopic discrimination of amino acids and fatty acids in polyps of the jellyfish Aurelia aurita in controlled feeding experiments, to understand the metabolic flux of these organic substrates in the jellyfish physiology under high-density populations. Contrary to the general trophic isotopic discrimination observed in non-gelatinous organisms, the discrimination in the A. aurita polyps is close to zero for most amino acids (except for glycine and leucine), indicating a little activity of both biosynthesis and degradation of the amino acids. In contrast, the discrimination in the A. aurita polyps is considerably large for fatty acids (ranging from 18.4‰ (for C 18:0 ) to 50.5‰ (for C 18:2 , n -6), indicating a large activity of degradation of fats (and its components, fatty acids). Based on these results, we conclude that A. aurita polyps prioritize the use of fatty acids compared to that of amino acids to produce life energy, for minimizing the production of nitrogenous waste derived from the amino acid degradation (about 0.0 mg/L for NH 3 ) and maintaining the quality of ambient water, and therefore for allowing them to survive in high-density populations. Moreover, these findings suggest the presence of metabolic plasticity among marine organisms. This plasticity may reflect flexible survival strategies supported by different metabolic pathways. Therefore, integrating the trophic isotopic discrimination of both amino acids and fatty acids can be useful for understanding these physiological mechanisms, as well as for accurately illustrating energy transfer along food chains in ecosystems. Graphical Abstract

Compound-specific isotope analysis (CSIA) of carbon and nitrogen within amino acids by gas chromatography/isotope ratio mass spectrometry (GC/IRMS) has been employed as a powerful tool for estimating the trophic tendency and resource utilization of organisms in food webs, as well as for illustrating the metabolic fate of amino acids and associated energy flux in diverse levels from within a single organism to entire ecosystems. However, the polar functional groups (i.e., carboxyl, amino, and hydroxyl groups) of amino acids should be neutralized by derivatization (i.e., esterification followed by acylation) before injection into a GC/IRMS instrument, which has caused several serious issues. For instance, the toxicity of acylation reagents (e.g., pivaloyl chloride) and the fluorinated derivatives (e.g., derived from trifluoroacetic anhydride) have complicated the use of isotope ratio analysis of amino acids in our studies. Moreover, large isotopic fractionation in 13 C/ 12 C associated with the acylation has considerably reduced the accuracy of the analysis of carbon isotope ratios. In the present study, we therefore developed a new derivatization method to minimize these issues, which can reduce the isotopic fractionation in 13 C/ 12 C by following two sequential reactions for the acylation: the first is acylation, which introduces isotopic fractionation, and the second is replacement of the pivaloyl group with that of pivalic anhydride. Further, the contribution of the 13 C/ 12 C ratios of derivative groups (i.e., isopropyl and pivaloyl groups) can be completely removed by a simultaneous mass balance calculation. Also, the new method is relatively safe, without the use of toxic reagents and the production of fluorinated derivatives. Thus, this derivatization method can be useful for CSIA of amino acids in diverse studies, which promises to provide unprecedented accuracy in understanding the complex physiological, ecological, and geochemical dynamics across aquatic and terrestrial ecosystems.

Nitrogen isotope ratios of amino acids (δ 15 N AA ) have widely been employed as a powerful tool for estimating the trophic position of organisms in food webs. This estimation is based on an elevation pattern in the δ 15 N AA value, a large elevation for the trophic amino acids (e.g., 8.0‰ for glutamic acid) while a little elevation for the source amino acids (e.g., 0.4‰ for phenylalanine), associated with the deamination of amino acids in consumers. However, several elevation patterns can be found in natural environments, one of which may be caused by the hydrolysis of specific protein under nutritional stress. In the present study, we identified ‘protein-specific elevation pattern’ for collagen, which connected to muscle tissues of fish and gastropods under nutritional stress. Time-series starvation for 45 days results in that collagen is consumed by 86% for the fish Girella   punctata and by 50% for the gastropod Turbo   sazae . Moreover, during the starvation, although a little change in the δ 15 N AA value is found in muscle fibers, a gradual elevation in the δ 15 N AA value is found in collagen for both trophic and source amino acids (e.g., by up to 11‰ for glutamic acid and up to 3‰ for phenylalanine, respectively, which can be explained by the Rayleigh fractionation model). We thus demonstrate that the consumption of collagen in organisms under starvation shows a unique elevation pattern in the δ 15 N AA value, which is consistent with the observation that collagen is degraded non-quantitatively by the collagenase reaction, whereas muscle is degraded quantitatively by the chaperone-mediated autophagy. The effect of δ 15 N elevation in all amino acids of collagen is negligible for G. punctata and diluted for T. sazae to the elevation in whole muscle tissue (i.e., = muscle fibers + collagen) even under long-term starvation, because the collagen proportion in the whole muscle is relatively small and is considerably decreased under the nutritional stress. Based on these results, we predict that the difference in the δ 15 N AA value between collagen and muscle fibers can be useful for evaluating the nutritional stress of fishes. However, great care will be required if studying food webs where collagen-rich organisms (i.e., gelatinous zooplankton) are abundant or if using collagen-rich materials in our studies.

MALDI Time-of-Flight Mass Spectrometry (MALDI-TOF MS) provides a fast and reliable alternative method for species-level identification of pathogens and various metazoans. Compared to the commonly used mitochondrial cytochrome c oxidase subunit I (mtCOI) barcoding, advantages of MALDI-TOF MS are rapid species identifications and low costs. In this study, we used MALDI-TOF MS to determine whether spectra patterns of different species can be used for species identification. We obtained a total of 138 spectra from individual specimens of Tigriopus , which were subsequently used for various cluster analyses. Our findings revealed these spectra form three clear clusters with high AU value support. This study validates the viability of MALDI-TOF MS as a methodology for higher-resolution species identification, allowing detection of cryptic species of harpacticoida. In addition, we propose a new species, Tigriopus koreanus sp. nov. by utilizing integrative methods such as morphological comparison, mtCOI barcoding, and MALDI-TOF MS.

Nitrogen isotope ratios of amino acids have been employed as a tool for illustrating the resource utilization of organisms and the trophic transfer between organisms in food webs. This tool is proposed based on the empirical observation that a universal trophic enrichment in 15 N is found in glutamic acid from diet to consumer species. Since glutamic acid is deaminated and incorporated into the tricarboxylic acid cycle (aerobic metabolism), the isotope ratios of glutamic acid have been used for evaluating life energy production through aerobic metabolism. However, little is known about the change in the isotope ratio associated with ‘anaerobic’ metabolism such as fermentation in organisms. Therefore, we determined the isotopic fractionation factor ( α ) for the deamination of alanine, a representative amino acid related to anaerobic metabolism, and conducted laboratory-cultured experiments to see the change in the nitrogen isotope ratio through anaerobic metabolism. Moreover, we compared the trophic enrichment in 15 N between alanine and glutamic acid in natural samples to visualize the contribution of anaerobic metabolism in food webs. In the in vitro enzymatic deamination of alanine, the isotope ratios of alanine are gradually increased, with the α of 0.9923. In the culture of the eukaryote Saccharomyces cerevisiae and the bacterium Lactococcus lactis with organic substrates under hypoxic conditions, the enrichment in 15 N is negligibly small for glutamic acid (by ~ 0‰), but considerably large for alanine (by ~ 5‰) in the culture. These results demonstrate that the activity of anaerobic metabolism is recorded in the isotope ratios of alanine but not glutamic acid in organisms. Moreover, we visualize the contribution of anaerobic metabolism in natural samples: (1) hypoxia within pollen provision for the diet of bee larvae induces a large enrichment in 15 N of alanine; and (2) this ‘anaerobic’ enrichment in 15 N can be identified in the cross-plot of the isotope ratios for alanine and glutamic acid in marine and terrestrial species, dissolved organic nitrogen, and the bees that potentially record of hypoxia within their diets. Thus, we suggest that the comparison of the isotope ratios between alanine and glutamic acid can be useful for evaluating the experience of hypoxia and the activity of anaerobic metabolism in biological and environmental samples.

Despite their abundance in marine ecosystems, studies on siphonophores are limited. In this study, 26 species of siphonophores in the Northwest Pacific Ocean were identified during multiple cruises of the R/V ISABU from 2018–2020, and various factors that may affect the occurrence of siphonophores, including water temperature, salinity, zooplankton biomass, and trophic niche were investigated. Statistical analysis revealed that the distribution of siphonophores and their biomass could be divided into two water mass groups, affected by the Kuroshio and Oyashio Currents. The species with high contributions to distinguishing the water mass groups (including Chelophyes contorta , Dimophyes arctica , Bassia bassensis , and Eudoxoides spiralis —mainly belonging to the Diphyidae) showed species-specific correlations with water temperature and salinity. This suggests that diphyids can be used as indicator species for currents and hydrological factors that influence water mass. The biomass of siphonophores exhibited a trend opposite to that of non-gelatinous zooplankton and showed no association with other gelatinous zooplankton. These results can be interpreted from an ecological niche perspective. Through nitrogen and carbon stable isotope analyses, the dietary sources of siphonophores could potentially overlap with those of chaetognaths or non-gelatinous zooplankton. Because the trophic position of siphonophores (2.4–3.2) also falls in the range of those of chaetognaths (2.8–3.4) and non-gelatinous zooplankton including copepods, euphausiids, and amphipods (2.4–3.5), diet competition with carnivorous mesozooplankton could be predicted. Considering that the diversity and biomass of most siphonophores are strongly positively correlated with water temperature and salinity, expansion of the Kuroshio Current is expected to lead to an increase in siphonophores in the Northwest Pacific in the future. The findings of this study are anticipated to provide novel insights into climate change prediction and response and enhance our understanding of siphonophore communities.

In recent years, the overuse of antibiotics has led to the emergence of antimicrobial-resistant (AMR) bacteria. To evaluate the spread of AMR bacteria, the reservoir of AMR genes (resistome) has been identified in environmental samples, hospital environments, and human populations, but the functional role of AMR bacteria and their persistence within individuals has not been fully investigated. Here, we performed a strain-resolved in-depth analysis of the resistome changes by reconstructing a large number of metagenome-assembled genomes from the gut microbiome of an antibiotic-treated individual. Interestingly, we identified two bacterial populations with different resistome profiles: extensively acquired antimicrobial-resistant bacteria (EARB) and sporadically acquired antimicrobial-resistant bacteria, and found that EARB showed broader drug resistance and a significant functional role in shaping individual microbiome composition after antibiotic treatment. Our findings of AMR bacteria would provide a new avenue for controlling the spread of AMR bacteria in the human community.

Conditioned pain modulation (CPM) and temporal summation (TS) tests can measure the ability to inhibit pain in fibromyalgia syndrome (FMS) patients and its level of pain sensitization, respectively. However, their clinical validity is still unclear. We studied the association between changes in the CPM and TS tests and the clinical improvement of FMS patients who received therapeutic intervention. We systematically searched for FMS randomized clinical trials with data on therapeutic interventions comparing clinical improvement (pain intensity and symptom severity reduction), CPM, and TS changes relative to control interventions. To study the relationship between TS/CPM and clinical measures, we performed a meta-regression analysis to calculate odds ratios. We included nine studies (484 participants). We found no significant changes in TS or CPM by studying all the interventions together. Our findings show that this lack of difference is likely because pharmacological and non-pharmacological interventions resulted in contrary effects. Non-pharmacological interventions, such as non-invasive neuromodulation, showed the largest effects normalizing CPM/TS. Meta-regression was significantly associated with pain reduction and symptom severity improvement with normalization of TS and CPM. We demonstrate an association between clinical improvement and TS/CPM normalization in FMS patients. Thus, the TS and CPM tests could be surrogate biomarkers in FMS management. Recovering defective endogenous pain modulation mechanisms by targeted non-pharmacological interventions may help establish long-term clinical recovery in FMS patients.