Multigene Phylogenies of the Estuarine Sesarmid Parasesarma bidens Species Complex (Decapoda: Brachyura: Sesarmidae), with Description of Three New Species.

S Parasesarma bidens (De Haan, 1835) is a common intertidal sesarmid crab that inhabits the estuarine and mangrove regions of East Asia, as well as Vietnam and the Philippines. Previous studies have suggested that P. bidens is a species complex composed of several species. Our findings indicated that the P. bidens species complex is monophyletic with six clades, as shown by reconstructed phylogenies based on mitochondrial 16S rDNA and cytochrome c oxidase subunit I, as well as nuclear 28S rDNA. Specimens of each clade exhibited different characters, including the morphology of the cheliped, anterior carapace, male gonopod 1, and female vulvae, as well as the coloration. Three species in this complex have been previously described: P. bidens (De Haan, 1835), P. cricotum (Rahayu & Davie, 2002), and P. sanguimanus Li, Shih & Ng, 2019. Here, we describe three additional species: P. continentale n. sp., P. insulare n. sp., and P. chiahsiang n. sp. Biogeographically, the range of P. bidens includes the southern margin of Korea, the main islands of Japan, and the Ryukyus, with a comparatively northern distribution. P. continentale is widely distributed from the continental coasts of southeastern and southern China (including northern Hainan Island) to southern Vietnam and is occasionally found in Penghu, Taiwan. Parasesarma insulare is an insular species distributed from Taiwan main island, the central Philippines, to northern Borneo. Finally, P. chiahsiang is mainly limited to the Penghu Islands (Pescadores) but is also sometimes found in Taiwan main island and the offshore Gueishan Island.

Taxonomy and Phylogeography of the Freshwater Crab Geothelphusa tawu Species Complex (Crustacea: Decapoda: Potamidae) from Southern Taiwan and Offshore Islets.

The freshwater crabs Geothelphusa tawu Shy, Ng & Yu, 1994, G. lutao Shy, Ng & Yu, 1994 and G. lanyu Shy, Ng & Yu, 1994 from southern Taiwan and the offshore islets, Lyudao (Green I.) and Lanyu (Orchid I.) are closely related in morphology and genetics, and have been proposed to be the same species. Examination of a series of specimens collected from the distributional ranges of the three species indicated that key characters of the ambulatory legs and the male first gonopod (G1) are too variable to support the identity of three species. Based on the mitochondrial 16S rDNA and cytochrome oxidase subunit I (COI) sequences, the phylogenetic analysis did not recover three clades corresponding to the three species, but only a main clade without further clear grouping. The interspecific distances of nucleotides are also too small to support the species delimitation. We conclude that the three species should be treated as a single species. Additionally, unique haplotypes of COI have been found in Lyudao and Lanyu, which are hypothesized as two founder populations that colonized the islets from the Taiwan main island during glacial maxima.

First Record of the Genus Pseudohelice Sakai, Türkay & Yang, 2006 from India and Description of a New Pseudocryptic Species (Crustacea: Brachyura: Varunidae).

A new pseudocryptic species of the varunid crab genus Pseudohelice Sakai, Türkay & Yang, 2006, is described from India based on morphological and molecular evidence. The new species was collected from higher intertidal zones in the Vellar River estuary, Tamil Nadu, southeastern India, in a habitat composed of muddy and sandy sediment. Pseudohelice annamalai n. sp. is similar to P. subquadrata (Dana, 1851) and P. latreillii (H. Milne Edwards, 1837) in general form, but can be distinguished from the congeners by the characters of the infraorbital ridges, male first gonopod, and female vulvae. In addition, the mitochondrial cytochrome oxidase subunit I sequences also support the new species. The occurrence of Pseudohelice from India links the distribution gap between the western Indian Ocean and western Pacific Ocean. The new species provides additional evidence for the geographic isolation of the eastern Indian Ocean for some marine organisms.

Di(2-ethylhexyl)phthalate exposure exacerbates metabolic disorders in diet-induced obese mice.

Both phthalate exposure and obesity are positively associated with metabolic disorders. The study aimed to investigate whether DEHP exposure caused metabolic disorders in an obesity-dependent manner. Both lean and diet-induced obese mice were subjected to environmentally relevant DEHP exposure. DEHP-treated obese mice exhibited higher glucose intolerance and insulin resistance than obese mice; the metabolic disorders were accompanied by increased blood levels of leptin, LDL cholesterol, and alanine transaminase. In obese mice, DEHP enhanced macrophage infiltration into epididymal white adipose tissue (eWAT) and hepatic tissue, and promoted hepatic steatosis/steatohepatitis. The DEHP effects were not observed in lean mice. Transcriptomic changes in eWAT and hepatic tissue were determined with microarray analysis. Results indicated that obesity and DEHP synergistically regulated carbohydrate uptake, lipolysis, and abnormality of adipose tissue, via the upstream regulators Pparg, Lipe, Cd44, and Irs1. Meanwhile, obesity and DEHP differentially modulated transcriptomic changes in hepatic tissue. Obesity was associated with lipid/cholesterol synthesis, lipid accumulation, and inflammation in hepatic tissue via the upstream regulators Zbtb20 and Nr1i2. In obese mice, DEHP exposure caused hepatic injury, cell migration, and changes in glycogen quantity mainly via Cd44. Microarray analysis suggested the potential mechanism underlying the early onset of metabolic disorders in DEHP-treated obese mice.

Phthalate exposure causes browning-like effects on adipocytes in vitro and in vivo.

Mono(2-ethylhexyl)phthalate (MEHP) promotes adipogenesis via PPARγ. PPARγ agonists, e.g., rosiglitazone (RSG), enhance adipocyte browning. However, scientific evidence regarding MEHP as a browning chemical is lacking. This study combined 3T3-L1 adipocytes and C57BL/6J mice to examine the potential roles of MEHP in browning. MEHP and the browning agent RSG caused similar energy metabolism in adipocytes. Both MEHP and RSG caused transcriptional changes involved in browning-associated thermogenesis, energy homeostasis, inflammatory response, and glucose uptake. MEHP-treated adipocytes exhibited brown adipocyte-like characteristics, i.e., increased mitochondrial proton leak, triiodothyronine-induced Bmp8b expression, decreased inflammation, and smaller lipid droplets. Increased PDK4 and PEPCK1 in MEHP/RSG-treated adipocytes could block glucose utilization for mitochondrial respiration. Mitochondrial/peroxisomal biogenesis and fatty acid ß-oxidation in MEHP-treated adipocytes were enhanced. Candidate genes in promoting browning of MEHP-treated adipocytes were highlighted. In di(2-ethylhexyl)phthalate (DEHP)-treated mice, transcriptional changes in white adipose tissue (WAT) were associated with adipocyte differentiation, lipid synthesis, carbohydrate uptake, and WAT/brown adipose tissue (BAT) quantity. PPARγ and NR4A1 were predicted as the top two upstream regulators in orchestrating transcriptional changes. DEHP-treated mice exhibited actively expressed browning marker genes (i.e., Pparg, Adrb1, Adrb3, Ppargc1a, and Ucp1) in WAT, increased blood FGF21 levels, and higher amounts of BAT, supporting the browning-like effects in vivo.

The identities of three species of Parahelice Sakai, Türkay amp; Yang, 2006 (Crustacea: Brachyura: Varunidae) from the western Pacific, based on morphological and molecular evidence.

Species of the varunid genus Parahelice Sakai, Türkay Yang, 2006, inhabit high intertidal areas of oceanic islands of the tropical Indo-West Pacific region. As several species of Parahelice and Pseudohelice subquadrata (Dana, 1851) were found to be sympatric in some places, and their morphological differences are minor, especially in females, the misidentification of species is not uncommon in the literature. In this study, the DNA barcoding marker, mitochondrial cytochrome c oxidase subunit I (COI), was applied to confirm species identities and this was correlated with the specific characters of males and females. Distributions of three species of Parahelice were also updated, with Par. daviei (Sakai, Türkay Yang, 2006), Par. pilimana (A. Milne-Edwards, 1873), and Par. pilosa (Sakai, Türkay Yang, 2006) being new records to Taiwan, and Par. pilosa new to Bali, Indonesia.

Diversity of Taiwanese Brackish Crabs Genus Ptychognathus Stimpson, 1858 (Crustacea: Brachyura: Varunidae) based on DNA Barcodes, with Descriptions of Two New Species.

Species in the brackish crab genus Ptychognathus are common in the seashore and estuary habitats with freshwater input. Due to their similar morphologies and dull colorations, it is always difficult to distinguish the species in this genus. In this study, the DNA barcode gene COI (cytochrome c oxidase subunit I) was used to help identify Ptychognathus from Taiwan. The results showed that the 10 species can be identified successfully based on COI, with intraspecific distances below 1.54% and interspecific distances of 12.2%-19.57%. In addition, two new species of Ptychognathus are described from Taiwan. Ptychognathus makii sp. nov. from southern Taiwan is similar to P. altimanus (Rathbun, 1914), and P. stimpsoni sp. nov. from southern Taiwan and the southern Philippines resembles P. aff. barbatus (A. Milne-Edwards, 1873) and P. pusillus Heller, 1865. Both species can be distinguished from other congeners by a suite of characters, including the carapace, orbital region, frontal region, telson of male pleon, male first gonopod, and setae on ambulatory legs.

Review of the mudflat varunid crab genus Metaplax (Crustacea, Brachyura, Varunidae) from East Asia and northern Vietnam.

Intertidal mudflat crabs of the genus Metaplax H. Milne Edwards, 1852 (Crustacea: Brachyura: Varunidae) from China, Taiwan, and northern Vietnam are taxonomically revised by morphological and molecular evidence. These crabs show sexual dimorphism and morphological variation of a considerable range in the infraorbital ridge, one of the primary features previously used for species identification. In this study, four species were identified from the region: M. elegans De Man, 1888; M. longipes Stimpson, 1858; M. sheni Gordon, 1930; and M. tredecim Tweedie, 1950. Based on the results of the morphological examination, and as confirmed by molecular evidence from mitochondrial cytochrome oxidase subunit I (COI), taxonomic confusion surrounding M. longipes was resolved, and M. takahasii Sakai, 1939, is considered a junior synonym of M. longipes. The geographical distribution of Metaplax longipes extends along the shores of China, north to Jiangsu, whereas the Southeast Asian M. tredecim was newly recorded from northern Vietnam and Hong Kong.

Involvement of metabotropic glutamate receptor 5 in ethanol regulation of NMDA receptor activity in rat substantia gelatinosa neurons.

AIMS: Glutamatergic receptors are important targets of ethanol. Intake of ethanol may produce analgesic effects. The present study examined the effects of ethanol on the activity of ionotropic glutamate receptors in spinal cord substantia gelatinosa (SG) neurons, critical neurons involved in nociceptive transmission. MAIN METHODS: Whole-cell recordings were made from SG neurons of the lumbar spinal cord slices from 15 to 20-day-old rats. Ethanol and glutamate receptor agonists or antagonists were applied by superfusion. KEY FINDING: Ethanol (50 and 100 mM) applied by superfusion for 5 min dose-dependently decreased the amplitude of evoked excitatory postsynaptic potential in SG neurons. Superfusion of ethanol (100 mM) for 15 min consistently inhibited NMDA- or AMPA-induced depolarizations in SG neurons. Ethanol (100 mM) also inhibited the depolarizations induced by glutamate. However, ethanol inhibition of glutamate-induced responses significantly decreased at 10-15 min following continuous superfusion, suggesting the development of acute tolerance to the inhibition during prolonged exposure. Application of MPEP hydrochloride (an antagonist of metabotropic glutamate receptor [mGluR] 5) or GF109203X (a protein kinase C [PKC] inhibitor), together with ethanol significantly blocked the tolerance. The inhibition by ethanol of the NMDA-induced, but not AMPA-induced, depolarizations significantly decreased at 15 min during continuous superfusion while ACPD (a mGluR agonist) was co-applied with ethanol. SIGNIFICANCE: The results suggest that (1) ethanol exposure may inhibit ionotropic glutamate receptor-mediated neurotransmission; (2) regulation of NMDA receptor function by mGluR5/PKC pathways may be involved in the development of the tolerance to ethanol inhibition of glutamate-induced responses during prolonged exposure in SG neurons.

Fibroblast growth factor 21 secretion enhances glucose uptake in mono(2-ethylhexyl)phthalate-treated adipocytes.

Previous studies revealed that cellular accumulation of mono(2-ethylhexyl)phthalate (MEHP) disturbed energy metabolism in adipocytes, where glucose uptake was significantly increased. The present study aimed to determine the mechanisms underlying the increased glucose uptake. MEHP-treated 3T3-L1 adipocytes exhibited a significantly increased glucose uptake activity. Immunoblot analysis suggested that the insulin-induced signals were not responsible for the increased glucose uptake. qPCR analysis revealed that both Glut1 and Glut4 genes were highly expressed during adipogenesis; Glut1 mRNA levels in MEHP-treated adipocytes were significantly increased. Moreover, MEHP-treated adipocytes exhibited significantly increased levels of fibroblast growth factor 21 (FGF21) in both mRNA and secreted protein. FGF21 is a peptide hormone with pleiotropic effects on regulation of insulin sensitivity and glucose/lipid homeostasis. We found that MEHP, FGF21, and lactate in culture medium together enhanced Fgf21 gene expression in MEHP-treated adipocytes. FGF21 signaling requires fibroblast growth factor receptor (FGFR) and ßKlotho. Fgfr family and ßKlotho genes were actively expressed during adipogenesis; mRNA levels of Fgfr3 and Fgfr4 genes in MEHP-treated adipocytes were significantly increased. Roles of FGF21/FGFR and phosphoinositide 3-kinase (PI3K)/AKT signal axes in regulation of glucose uptake were determined. We demonstrated that FGF21/FGFR signals played the major roles in up-regulation of the basal glucose uptake in MEHP-treated adipocytes. The in vitro evidence suggests that cellular FGF21 secretion enhances the basal glucose uptake in MEHP-treated adipocytes.

Estrogenic chemicals at body burden levels attenuate energy metabolism in 3T3-L1 adipocytes.

The study aimed to examine effects of environmental estrogens at body burden levels on energy metabolism in fat cells. Acclimation of T47D-KBluc cells in estrogen-deprived medium was established for high performance of estrogen-responsive luciferase reporter assay. With the assay, relative estrogenic potency of four selected estrogen receptor (ER) agonists, i.e. diethylstilbestrol, ß-estradiol, 4-nonylphenol and bisphenol A, were determined. Immunoblot analysis revealed that the ER agonists at both EC80 and EC100 caused rapid and transient phosphorylation of extracellular signal-regulated kinases (ERK) in an ER-dependent manner. 3T3-L1 adipocytes treated with the ER agonists at EC80 for 24 hours exhibited significant downregulation in mitochondrial respiration and glycolytic function. Importantly, EC80 values of 4-nonylphenol (6.0 × 10-10  m) and bisphenol A (1.0 × 10-8  m) are in the range of human body burdens. The finding that estrogenic chemicals at body burden levels cause significant impact on fat cell energy metabolism raises an important public health issue that deserves more attention.