Multigenerational resilience of the marine rotifer Brachionus plicatilis to high temperature after additive exposure to high salinity and nanoplastics.

To study multigenerational resilience to high temperature (HT) conditions, we exposed Brachionus plicatilis marine rotifers to HT, high salinity (HS), and nanoplastics (NPs), and measured reproductive and life-cycle endpoints. After exposure to HT, rotifer lifespans were reduced, but daily production of offspring increased. However, both combined HT/HS and HT/HS/NP exposure led to additional decreases in longevity and reproductive ability; the antioxidant defense mechanisms of the rotifers were also notably upregulated as measured by reactive oxygen species levels. Fatty-acid profiles were reduced in all conditions. In multigenerational experiments, the negative effects of HT dissipated rapidly; however, the effects of HT/HS and HT/HS/NPs required four generations to disappear completely. The findings indicated that B. plicatilis were able to recover from these environmental stressors. This study demonstrated the resilience of aquatic organisms in response to changing environmental conditions and provides insights into the complex interactions of different abiotic stressors.

Effects of bisphenol A on reproduction, oxidative stress, and lipid regulation in the marine rotifer Brachionus plicatilis.

This study reports the effects of bisphenol A (BPA) on the rotifer Brachionus plicatilis, focusing on growth performance, reproductive output, oxidative stress responses, and lipid metabolism genes. High BPA levels disrupted peak daily offspring production and led to oxidative stress and increased superoxide dismutase and catalase activity. The research identified distinctive monoacylglycerol O-acyltransferase (MGAT) and diacylglycerol O-acyltransferase (DGAT) genes in B. plicatilis, B. rotundiformis, and B. koreanus, enhancing understanding of lipid metabolism in these species. BPA exposure significantly altered MGAT and DGAT expression, and feeding status affected these regulatory patterns. When food was unavailable, BPA reduced DGAT2 and MGAT2a expression. However, under feeding conditions, DGAT2 and MGAT1 levels increased, indicating that nutritional status and BPA exposure interact to affect gene expression.

Effects of temperature and combinational exposures on lipid metabolism in aquatic invertebrates.

Studies of changes in fatty acids in response to environmental temperature changes have been conducted in many species, particularly mammals. However, few studies have considered aquatic invertebrates, even though they are particularly vulnerable to changes in environmental temperature. In this review, we summarize the process by which animals synthesize common fatty acids and point out differences between the fatty acid profiles of vertebrates and those of aquatic invertebrates. Unlike vertebrates, some aquatic invertebrates can directly synthesize polyunsaturated fatty acids (PUFAs), which can be used to respond to temperature changes. Various studies have shown that aquatic invertebrates increase the degree of saturation in their fatty acids through an increase in saturated fatty acid production or a decrease in PUFAs as the temperature increases. In addition, we summarize recent studies that have examined the complex effects of temperature and combinational stressors to determine whether the degree of saturation in aquatic invertebrates is influenced by other factors. The combined effects of carbon dioxide partial pressure, food quality, starvation, salinity, and chemical exposures have been confirmed, and fatty acid profile changes in response to high temperature were greater than those from combinational stressors.

Genome-wide identification of fatty acid synthesis genes, fatty acid profiles, and life parameters in two freshwater water flea Daphnia magna strains.

The freshwater water flea Daphnia magna is a planktonic animal belonging to the Cladocera. To evaluate differences between two D. magna strains (KIT and NIES) in terms of life parameters and fatty acid profiles, we examined several endpoints. In the D. magna KIT strain, the numbers of total and cumulative offspring were lower at 23 °C and higher at 14 °C than in the D. magna NIES strain. However, at 14 °C, the D. magna KIT strain showed an increased lifespan. Although the n-3/n-6 polyunsaturated fatty acids (PUFA) ratio was always decreased at a low temperature, the PUFA ratio in the KIT strain had a higher value on day 3 than the NIES strain, which gave it higher adaptability to low temperature. In addition, we identified the elongation of very long chain fatty acids (elovl) and fatty acid desaturase (fad) genes, which are involved in fatty acid biosynthesis pathways, in the genomes of both D. magna KIT and NIES. The Elovl and Fad genes in both D. magna strains were highly conserved, including tandem duplicated Elovl 1/7 genes. This study provides new information about the molecular basis for the difference in temperature sensitivity between two strains of D. magna.

Combined exposure to microplastics and zinc produces sex-specific responses in the water flea Daphnia magna.

Microplastics are ubiquitous environmental pollutants and a great threat to the aquatic environment. Due to their small size (ranging from 1 µm to 5 mm), microplastics be easily ingested by a wide range of organisms and can serve as a vector for various contaminants. In this study, additive or possible synergistic effects of microplastics and zinc were demonstrated through sex-specific alterations in behavior, redox status, and modulation of detoxification-related genes in Daphnia magna, with males being more sensitive than females with stronger modulations of antioxidant responses, particularly on glutathione S-transferases expressions. Furthermore, we demonstrated microplastics may act as vectors for metals (Zn2+) in the aquatic environment in D. magna, with reduced bio-concentration of the total Zn concentration, inducing greater toxicity. Our findings demonstrated synergistic toxicity of the heavy metal Zn and microplastics and could contribute to greater understanding of sex-specific effects of microplastics in aquatic organisms.

Alleviation of tributyltin-induced toxicity by diet and microplastics in the marine rotifer Brachionus koreanus.

To determine the effects of tributyltin (TBT) upon multiple exposures of diet and microplastic in rotifer, in vivo life parameters were measured. In 10 µg/L TBT-exposed rotifer, the 1 and 0.5 x diet groups resulted in reproduction reduction. However, 10 x diet treatment showed no significant changes in the total fecundity, despite a decrease in daily reproduction. Besides, differences in the lifespan were observed in response to different diet regimens. TBT and/or MP-exposed parental rotifer (F0) showed a significant delay in the pre-reproductive day under 0.5 x diet regimen. In all dietary regimens, exposure to TBT and MP induced an increase in reactive oxygen species, but antioxidant activities were perturbed. To further verify the carryover effect of TBT toxicity, progeny rotifer (F1) obtained from 24 h TBT and/or MP-exposed F0 was used. Interestingly, the faster hatching rate was observed only in F1 obtained from 1 x diet regimen-exposed F0. However, in the 0.5 x diet, the total fecundity was reduced and the pattern of the daily reproduction was collapsed. Thus, the toxicity of TBT can be alleviated by MP and nutrition status, but TBT-induced toxicity and its carryover effect are inevitable.

Effects of low temperature on longevity and lipid metabolism in the marine rotifer Brachionus koreanus.

Low-temperature exposure prolongs lifespans and changes lipid metabolism but the relationship between longevity and lipids is largely unknown. Here, we examine the relationship between longevity and lipid metabolism at low temperatures (20 °C and 15 °C) compared with a 25 °C control. Life parameters, fatty acid composition, and transcriptome changes were analyzed in the monogonont rotifer Brachionus koreanus. In vivo life-parameter data indicate that lifespan and fecundity exhibit opposite correlations at low temperatures. The amount of total fatty acids decreased significantly at low temperatures but areas stained with Nile red increased at 15 °C compared with the control. From RNA-seq-based transcriptional analysis, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway-enrichment analysis were conducted. GO analysis shows that telomeres were positively regulated at low temperatures. KEGG pathway-enrichment results indicate that gene expression involved in lipid metabolism was activated with increased glycerol and/or choline synthesis at low temperatures. We suggest that reduced reproductive rates are associated with a decrease of lecithin, which is involved in the conversion of glycerol to triacylglycerol in response to low temperatures by lowering the temperature of body fluid.

An improved genome assembly and annotation of the Antarctic copepod Tigriopus kingsejongensis and comparison of fatty acid metabolism between T. kingsejongensis and the temperate copepod T. japonicus.

Copepods in the genus Tigriopus are widely distributed in the intertidal zone worldwide. To assess differences in fatty acid (FA) metabolism among congeneric species in this genus inhabiting polar and temperate environments, we analyzed and compared FA profiles of the Antarctic copepod Tigriopus kingsejongensis and the temperate copepod T. japonicus. Higher amounts of total FAs were found in the Antarctic copepod T. kingsejongensis than the temperate copepod T. japonicus under administration of the identical amount of Tetraselmis suecica. To determine the genomic basis for this, we identified fatty acid metabolism-related genes in an improved genome of T. kingsejongensis. The total length of the assembled genome was approximately 338 Mb with N50 = 1.473 Mb, 938 scaffolds, and a complete Benchmarking Universal Single-Copy Orthologs value of 95.8%. A total of 25,470 genes were annotated using newly established pipeline. We identified eight elongation of very long-chain fatty acid protein (Elovl) genes and nine fatty acid desaturase (Fad) genes in the genome of T. kingsejongensis. In addition, fatty acid profiling suggested that the duplicated Δ5/6 desaturase gene in T. kingsejongensis is likely to play an essential role in synthesis of different FAs in T. kingsejongensis to those in T. japonicus. However, further experimental research is required to validate our in silico findings. This study provides a better understanding of fatty acid metabolism in the Antarctic copepod T. kingsejongensis.

Effects of atrazine on life parameters, oxidative stress, and ecdysteroid biosynthetic pathway in the marine copepod Tigriopus japonicus.

Atrazine is a widely used pesticide which acts as an endocrine disruptor in various organisms. The aim of this study was to investigate adverse effects of atrazine on life parameters, oxidative stress, and ecdysteroid biosynthetic pathway in the marine copepod Tigriopus japonicus. In T. japonicus, no mortality was shown in response to atrazine up to 20 mg/L in acute toxicity assessment. In nauplii, retardation in the growth and prolonged molting and metamorphosis resulted under chronic exposure of atrazine at 20 mg/L. In addition, body sizes of T. japonicus nauplii were significantly decreased (P < 0.01 in length and P < 0.001 in width) in response to 20 mg/L of atrazine. Furthermore, atrazine induced oxidative stress by the generation of reactive oxygen species at all concentrations compared to the control in the nauplii. Also, significant increase in glutathione-S transferase activity was observed in adult T. japonicus at low concentration of atrazine. To understand effects of atrazine on ecdysteroid biosynthetic pathway-involved genes (e.g., neverland, CYP307E1, CYP306A1, CYP302A1, CYP3022A1 [CYP315A1], CYP314A1, and CYP18D1) were examined with mRNA expressions of ecdysone receptor (EcR) and ultraspiracle (USP) in response to 20 mg/L atrazine in nauplii and adults. In the nauplii, these genes were significantly downregulated (P < 0.05) in response to atrazine, compared to the control but not in the adult T. japonicus. These results suggest that atrazine can interfere in vivo life parameters by oxidative stress-induced retrogression and ecdysteroid biosynthetic pathway in this species.

Tributyltin Affects Retinoid X Receptor-Mediated Lipid Metabolism in the Marine Rotifer Brachionus koreanus.

To examine how tributyltin (TBT), a model obesogen, affects the lipid metabolism in the marine rotifer Brachionus koreanus, we carried out life-cycle studies and determined the in vitro and in silico interactions of retinoid X receptor (RXR) with TBT, the transcriptional levels of RXR and lipid metabolic genes, and the fatty acid content. The lethal concentration 10% (LC10) was determined to be 5.12 µg/L TBT, and negative effects on ecologically relevant end points (e.g., decreased lifespan and fecundity) were detected at 5 µg/L TBT. On the basis of these findings, subsequent experiments were conducted below 1 µg/L TBT, which did not show any negative effects on ecologically relevant end points in B. koreanus. Nile red staining analysis showed that after exposure to 1 µg/L TBT, B. koreanus stored neutral lipids and had significantly increased transcriptional levels of RXR and lipid metabolism-related genes compared to the control. However, the content of total fatty acids did not significantly change at any exposure level. In the single fatty acids profile, a significant increase in saturated fatty acids (SFAs) 14:0 and 20:0 was observed, but the contents of omega-3 and omega-6 fatty acids were significantly decreased. Also, a transactivation assay of TBT with RXR showed that TBT is an agonist of Bk-RXR with a similar fold-induction to the positive control. Taken together, these results demonstrate that TBT-modulated RXR signaling leads to increase in transcriptional levels of lipid metabolism-related genes and the synthesis of SFAs but decreases the content of polyunsaturated fatty acids (PUFAs). Our findings support a wider taxonomic scope of lipid perturbation due to xenobiotic exposure that occurs via NRs in aquatic animals.

Adverse effects, expression of defense-related genes, and oxidative stress-induced MAPK pathway in the benzo[α]pyrene-exposed rotifer Brachionus rotundiformis.

To examine the adverse effects of the benzo[α]pyrene (B[α]P), the monogonont rotifer Brachionus rotundiformis was exposed to various concentration of B[α]P (0 [control], 1, 10, and 100 µg/L) and measured life cycle parameters (e.g., mortality, fecundity [cumulated number of offspring], and lifespan), reactive oxygen species (ROS), antioxidant enzymatic activity of glutathione S-transferase (GST). In addition, defense-related transcripts (e.g., glutathione S-transferases [GSTs], ATP binding cassette [ABCs] transporters) and Western blot analysis of mitogen-activated protein kinase (MAPK) signaling pathway were investigated in B[α]P-exposed rotifer. In this study, the total intracellular ROS level and GST activity were significantly increased (P < 0.05), while fecundity and lifespan were also significantly (P < 0.05) reduced in a concentration dependent manner in B[α]P-exposed B. rotundiformis. In addition, transcriptional regulation of GSTs and ABC transporters were significantly upregulated and downregulated (P < 0.05), respectively, suggesting that B[α]P can induce oxidative stress leading to induction of antioxidant system and detoxification mechanism. In addition to detoxification-related genes, B[α]P-exposed B. rotundiformis showed the increased levels of the p-JNK and p-p38, suggesting that B[α]P can activate MAPK signaling pathway in B. rotundiformis.

Genome-wide characterization and expression of the elongation of very long chain fatty acid (Elovl) genes and fatty acid profiles in the alga (Tetraselmis suecica) fed marine rotifer Brachionus koreanus.

To understand the lipid metabolism in invertebrate species, identification of the fatty acid (FA) synthesis gene families in invertebrate species is important, since some FA are unable to be synthesized in the organisms by themselves. In the study, to identify the elongation of very long chain fatty acid (Elovl) genes in the marine rotifer Brachionus koreanus, the genome-wide identification and phylogenetic analysis of Elovl genes have been conducted with the expression profile of Elovl genes on the alga Tetraslemis suecica-fed B. koreanus. A total 10 Elovl genes have been identified from the genome of B. koreanus, with conserved HXXHH motif. Synteny analysis showed that tandem duplication event has occurred (Elovl3/6a and b, Elovl9a and b, and Elovl9c and d) in the ancestor. Phylogenetic analysis have clearly revealed that Brachionus spp. has only 2/5 and 3/6 subfamilies, and two novel Elovl classes have been revealed, namely Elovl9 and 10. Transcriptional data showed that the 10 Elovl genes were differently expressed and their expression could be regulated by feeding the alga T. suecica. From fatty acid (FA) profile data of the alga Tetraslemis suecica-fed B. koreanus, we revealed that the marine rotifer B. koreanus may synthesize very long chain fatty acid (VLCFA; >22 carbons) by themselves, as VLCFA was hardly detected in the alga T. suecica. The study provides a better understanding of FA metabolism of the marine rotifer B. koreanus after feeding the T. suecica.

Genome-wide identification and expression of the entire 52 glutathione S-transferase (GST) subfamily genes in the Cu2+-exposed marine copepods Tigriopus japonicus and Paracyclopina nana.

In this study, the entire glutathione S-transferases (GSTs), the major phase II detoxification enzyme, were identified in two marine copepod species Tigriopus japonicus and Paracyclopina nana. The genome-wide identification of GSTs in T. japonicus and P. nana resulted in 32 and 20 GSTs in total, respectively. Among the identified GSTs, two specific classes of GSTs, specifically sigma and delta/epsilon GSTs were the dominant form of cytosolic GSTs in T. japonicus, while delta/epsilon and mu classes were dominant cytosolic GSTs in P. nana. In addition, Membrane-Associated Proteins in Eicosanoid and Glutathione metabolism (MAPEG) family were found in relatively higher proportion compared to other classes. Moreover, sigma, delta/epsilon, and microsomal GSTs have shown to expand through tandem duplication. To validate the detoxification function of the identified GSTs, both copepods were exposed to copper (Cu2+) and the reactive oxygen species (ROS) level and GST activity were measured. With integration of phylogenetic analysis and xenobiotic-mediated GST mRNA expression patterns along with previous enzymatic activities, the functional divergence among species-specific GST genes was clearly observed. This study covers full identification of GST classes in two marine copepod species and their important role in marine environmental ecotoxicology.

Adverse effects of two pharmaceuticals acetaminophen and oxytetracycline on life cycle parameters, oxidative stress, and defensome system in the marine rotifer Brachionus rotundiformis.

To investigate the adverse effect of two widely used pharmaceuticals, paracetamol (acetaminophen [APAP]) and oxytetracycline (OTC) on the marine rotifer Brachionus rotundiformis (B. rotundiformis), the animals were exposed to various environmentally-relevant concentrations. Up to date, acetaminophen and oxytetracycline have been considered as toxic, if used above threshold concentration, i.e. overdosed. However, this study demonstrated these two pharmaceuticals even at low concentration (i.e., µg/L scale) elicited oxidative stress through the generation of reactive oxygen species (ROS) along with the increased glutathione S-transferase activity, despite no-observed effect in in-vivo population growth. To validate the adverse effects of the two pharmaceuticals at relatively low concentrations, mRNA expression analysis was performed of the entire set of genes encoding 26 cytochrome P450s (CYPs) of phase I and 19 glutathione S-transferases (GSTs) of phase II of the rotifer B. rotundiformis. The mRNA expression analysis suggested specific genes CYP3045A2 and GSTσ1, GSTσ4, and GSTω1 take part in detoxification of APAP and OTC, resulting in no significant changes in the population growth and undetermined no observed effect concentration (NOEC) in the marine rotifer B. rotundiformis.

Effects of bisphenol A and its analogs bisphenol F and S on life parameters, antioxidant system, and response of defensome in the marine rotifer Brachionus koreanus.

To understand the adverse outcome in response to bisphenol A and its analogs bisphenol F and S (BPA, BPF, and BPS), we examined acute toxicity, life parameter, and defensome in the marine rotifer Brachionus koreanus. Among the bisphenol analogs, BPA showed the highest acute toxicity and then BPF and BPS, accordingly in the view of descending magnitude of toxicity. In life parameters including life span and reproduction, BPA, BPF, and BPS were found to cause adverse effect. Both intracellular ROS level and GST activity were significantly increased (P < 0.05) in response to each dosage of bisphenol analogs exposures. In response to bisphenol analogs, defensomes of phase I, II, and III detoxification mechanism demonstrated inverse relationship between the lipophilicity of bisphenol analogs and the expression patterns of defensomes. BPA and BPF were found to have significant modulation (P < 0.05) in the expression of cytochrome P450 (CYP) and GST genes. In phase III, BPS with comparatively lower lipophilicity demonstrated highly diversified expressional pattern, suggesting that BPS is likely caused less toxicity compared to BPA and BPF. In this study, via phase I, II, and III detoxification mechanism, bisphenol A and its analogs F and S demonstrated specific detoxification mechanism in rotifer.

Aging extension and modifications of lipid metabolism in the monogonont rotifer Brachionus koreanus under chronic caloric restriction.

To examine the interrelationship of aging extension and modification of lipid metabolism under chronic caloric restriction (CCR; reduced concentration of the green algae Tetraselmis suecica) in the monogonont rotifer Brachionus koreanus, we assessed life cycle parameters, fatty acid composition, and expression of sirtuin and genes related to lipid metabolism. B. koreanus in the 5% T. suecica group showed an increased life span but decreased reproduction. Based on this finding, we chose 5% T. suecica for further experiments and compared the data with those for 100% T. suecica. Upregulation of sirtuin gene expression was observed under CCR. In addition, despite the reduction in the amount of total fatty acid (FA) and the area of triacylglycerol, increases in the ratios of saturated fatty acid and monounsaturated fatty acid (MUFA) to total FA in 5%-exposed B. koreanus were observed. Furthermore, mRNA expression analysis confirmed that CCR promoted the synthesis of MUFA through Δ9 desaturase. Moreover, expression of the docosahexaenoic acid (DHA) synthesizing gene Δ4 desaturase was also upregulated, together with DHA content. These data suggest that CCR modified protein acetylation and lipid metabolism, leading to a decrease in reproduction and consequently resulting in life span extension.