A Simplified Gas Chromatographic Fatty-Acid Analysis by the Direct Saponification/Methylation Procedure and Its Application on Wild Tuna Larvae

A method for the direct preparation of fatty‐acid methyl esters (FAME) was simplified for fatty‐acid analysis of a single fish larva using gas chromatography (GC). The method included the isolation of a larval trunk and drying in a glass vial, followed by saponification of all the contents without prior lipid extraction. Thereafter, the fatty acids released were methylated by trimethylsilyldiazomethane. This method has advantages over another method, direct acid‐catalyzed transesterification, because both the saponification and methylation at room temperature can reduce loss of unsaturated fatty acids and formation of artifacts unavoidable in acidic reaction at high temperature. GC of the products showed that the simplified method can yield methyl esters without artifacts interfering analysis. More than 50 fatty acids were determined, which are twice as many as those previously analyzed using high‐performance liquid chromatography. Observation of consistent small impurities in GC of blank tests allowed the accurate determination of fatty acids by correcting the peak areas. Dry matter weights (<3 mg) and the total fatty‐acid contents displayed a linear relationship. Fatty‐acid analysis of wild larvae of bluefin tuna, yellowfin tuna, and skipjack tuna collected from the waters around Japan (n = 100) revealed that the eicosapentaenoic acid (EPA) level in bluefin tuna collected from the Japan Sea was significantly higher than that in the three species collected from Nansei Islands. The simplified direct saponification/methylation method will be a powerful tool for investigating growth and survival of individual larval tuna and other fish species

Geographic, seasonal and ontogenetic variations of δ15N and δ13C of Japanese sardine explained by baseline variations and diverse fish movements

&lt;p&gt;&lt;span&gt;Understanding and predicting variability in the stable isotope ratios of nitrogen and carbon (δ15N and δ13C, respectively) of small pelagic fish is crucial to enable isotopic studies of a variety of marine predators that feed on them. However, because the isotope ratios reflect plastic feeding habits and fish migration in addition to baseline variation, their predictions require a mechanistic understanding of how each factor contributes. Here, we investigated the habitat-wide variability of δ&lt;sup&gt;15&lt;/sup&gt;N and δ&lt;sup&gt;13&lt;/sup&gt;C of the Japanese sardine &lt;em&gt;Sardinops melanostictus&lt;/em&gt; in the western North Pacific and its marginal seas (the East China Sea and the Sea of Japan). By combining this with the archived particulate organic matter (POM) dataset as a baseline, we aimed to understand how ecological processes and baseline fluctuations affect isotope ratios of the sardine. Both δ&lt;sup&gt;15&lt;/sup&gt;N and δ&lt;sup&gt;13&lt;/sup&gt;C of sardine showed significant geographical and seasonal trends, with higher values in southern nearshore areas, including the Seto Inland Sea, intermediate values in marginal seas and lower values in Pacific offshore areas. As the variations were largely consistent with the geographic and temporally integrated seasonal trends of isotope ratios of POM, respectively, the baseline variations are the main determinant of sardine isotope composition. The trophic levels of sardine are therefore not significantly different between regions, with possible minor increases in the southern nearshore area. Adults showed less geographic variation than larvae and juveniles, likely due to slower turnover periods and wider migration ranges. Although larval and juvenile isotope ratios in marginal seas mostly reflected the local baseline, those in the Pacific offshore often reflected the baseline in the neighbouring southern region, suggesting contrasting juvenile movements between regions. Our results suggest that the δ&lt;sup&gt;15&lt;/sup&gt;N and δ&lt;sup&gt;13&lt;/sup&gt;C of Japanese sardine strongly reflect baseline variations, but can also be influenced by life-stage- and region-dependent fish movements, thereby demonstrating both the possibility and difficulty of mechanistically modelling the isoscapes of lower trophic level species.&lt;/span&gt;&lt;/p&gt;&lt;p&gt;Funding provided by: Ministry of Agriculture, Forestry and Fisheries&lt;br&gt;Crossref Funder Registry ID: https://ror.org/02zdz1m23&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;Funding provided by: Fisheries Research Agency&lt;br&gt;Crossref Funder Registry ID: https://ror.org/02gmwvg31&lt;br&gt;Award Number: &lt;/p&gt;&lt;p&gt;&lt;span&gt;Tissues were freeze-dried and ground into powder. Lipids were extracted from all samples using a 2:1 chloroform:methanol solution, freeze-dried again, and 800 μg of a subsample was extracted for isotope analysis. The δ&lt;sup&gt;15&lt;/sup&gt;N and δ&lt;sup&gt;13&lt;/sup&gt;C values of the samples were determined at Fisheries Resources Institute (Yokohama, Japan) or GeoScience Laboratory (Nagoya, Japan) using a continuous-flow stable isotope ratio mass spectrometer (IsoPrime100, Elementar, Stockton, UK; Delta Plus Advantage, Thermo Fisher Scientific, Waltham, Massachusetts, USA) coupled to an elemental analyser (vario MICRO cube, Elementar; FLASH2000, Thermo Fisher Scientific, Yokohama Japan). The δ&lt;sup&gt;15&lt;/sup&gt;N and δ&lt;sup&gt;13&lt;/sup&gt;C values were reported in δ-notation against the atmospheric N2 standard and the VPDB reference standard (Vienna Pee Dee Belemnite), respectively, and given as a ‰ value. Analytical accuracies were ± 0.2‰ for δ&lt;sup&gt;15&lt;/sup&gt;N and δ&lt;sup&gt;13&lt;/sup&gt;C in both laboratories. The agreement of the reported values between the two laboratories was tested using a blind standard (powder of fish eye lens) where the differences in the reported values for both δ&lt;sup&gt;15&lt;/sup&gt;N and δ&lt;sup&gt;13&lt;/sup&gt;C were less than the analytical precisions.&lt;/span&gt;&lt;/p&gt

Pelagic larval duration, growth rate, and population genetic structure of the tidepool snake moray Uropterygius micropterus around the southern Ryukyu Islands, Taiwan, and the central Philippines

The relationships between pelagic larval duration (PLD) and geographic distribution patterns or population genetic structures of fishes remain obscure and highly variable among species. To further understand the early life history of the tidepool snake moray Uropterygius micropterus and the potential relationship between PLD and population genetic structure of this species, otolith microstructure and population genetics based on concatenated mtDNA sequence (cytochrome b and cytochrome oxidase subunit I, 1,336 bp) were analyzed for 195 specimens collected from eight locations around the southern Ryukyu Islands, Taiwan, and the central Philippines. Eels with longer PLD and lower otolith growth rates were observed at relatively higher latitudes with lower water temperatures (54.6 ± 7.7 days and 1.28 ± 0.16 µm day−1 on Ishigaki Island, Japan, vs. 43.9 ± 4.9 days and 1.60 ± 0.19 µm day−1 on Badian, the Philippines), suggesting that leptocephali grew faster and had shortened pelagic periods in warmer waters. Meanwhile, the eels along the southwest coast of Taiwan had relatively longer PLD (57.9 ± 10.5 days), which might be associated with the more complex ocean current systems compared to their counterparts collected along the east coast of Taiwan (52.6 ± 8.0 days). However, the southwestern and eastern Taiwan groups had similar otolith growth rates (1.33 ± 0.19 µm day−1 vs. 1.36 ± 0.16 µm day−1). Despite the intergroup variation in PLD, genetic analysis revealed fluent gene flow among the tidepool snake morays in the study regions, implying that intraspecies PLD variation had a weak effect on genetic structure. The leptocephalus stage might have ensured the widespread gene flow among the study areas and leptocephalus growth was likely influenced by regional water temperature

Duality of n-3 Polyunsaturated Fatty Acids on Mcp-1 Expression in Vascular Smooth Muscle: A Potential Role of 4-Hydroxy Hexenal

N-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have protective effects against atherosclerosis. Monocyte chemotactic protein (MCP)-1 is a major inflammatory mediator in the progression of atherosclerosis. However, little is known about the regulation of Mcp-1 by DHA and EPA in vessels and vascular smooth muscle cells (VSMCs). In this study, we compared the effect of DHA and EPA on the expression of Mcp-1 in rat arterial strips and rat VSMCs. DHA, but not EPA, suppressed Mcp-1 expression in arterial strips. Furthermore, DHA generated 4-hydroxy hexenal (4-HHE), an end product of n-3 polyunsaturated fatty acids (PUFAs), in arterial strips as measured by liquid chromatography-tandem mass spectrometry. In addition, 4-HHE treatment suppressed Mcp-1 expression in arterial strips, suggesting 4-HHE derived from DHA may be involved in the mechanism of this phenomenon. In contrast, Mcp-1 expression was stimulated by DHA, EPA and 4-HHE through p38 kinase and the Keap1-Nuclear factor erythroid-derived 2-like 2 (Nrf2) pathway in VSMCs. In conclusion, there is a dual effect of n-3 PUFAs on the regulation of Mcp-1 expression. Further study is necessary to elucidate the pathological role of this phenomenon