ACUTE NITRATE EXPOSURE CAUSES PROTEOMIC CHANGES CONSISTENT WITH THE REGULATION OF REACTIVE OXYGEN AND NITROGEN SPECIES

Nitrate is the most common ionic form of nitrogen in aquatic ecosystems. Although nitrate is known to affect ecosystems at high levels through eutrophication, hypoxia and loss of biodiversity, it is considered to be physiologically inert to the individual aquatic organism. To test the physiological effects of nitrate on aquatic life, we exposed gill tissue of the Pacific oyster, Crassostrea gigas, to nitrate and characterized changes in protein expression, using a gel-based proteomics approach. Of the 642 protein spots detected, we found that 24 proteins (15 identified) changed expression in response to a 6-hour exposure to nitrate concentrations ranging from 0-73 mg/L, values that characterize highly contaminated surface and ground waters. Proteins changing expression included the oxidative stress proteins thioredoxin and cavortin (a member of the superoxide dismutase family) as well as proteins that are involved in G-protein signaling (Rho-GDI, ADP-ribosylation factor, G-protein ß-subunit), protein homeostasis (heat shock protein 70, prohibitin, calreticulin, and proteasome &#;-type 4 subunit), glycolysis (enolase), transport of hydrophobic molecules (lipocalin) and cytoskeletal arrangements (intermediate filaments and a gelsolin-like adseverin). The most parsimonious explanation for these changes in protein expression assumes that C. gigas reduces nitrate to nitrite and nitric oxide, which reacts with superoxide anions to form the very reactive peroxynitrite. We propose that part of the cellular response to reactive nitrogen species,phagocytic hemocytes inhibit the production of reactive oxygen species, potentially compromising the immune response of oysters to invading pathogens

The Proteomic Response of Gill Tissue in Tidally and Subtidally-Acclimated California Mussels, Mytilus californianus, to Acute Emersion-Induced Anoxia

Intertidal mussels regularly experience emersion-induced anoxia, in contrast to normoxic conditions experienced during submersion. We therefore hypothesized that acclimation to a tidal rhythm, as opposed to a rhythm of constant submersion, preconditions the proteome of the California mussel, Mytilus californianus, to respond differently to emersion-induced anoxia. Following acclimation, mussels either continued to receive the acclimation conditions (control) or were exposed to 100% nitrogengas (anoxia) during aerial emersion. We collected gill tissue for subsequent analysis of protein abundance with 2D gel electrophoresis and protein identification with tandem mass spectrometry. Relative to subtidally-acclimated mussels, tidally-acclimated mussels showed a greater propensity to respond to distrupted protein homeostasis during emersion through higher levels of several small heat shock protein isoforms, while they showed lower levels of several chaperones involved in redox-sensitive protein maturation in the endoplasmic reticulum during acute anoxia. Several metabolic proteins showed elevated levels in tidally-acclimated mussels, suggesting a compensatory response to reduced feeding times. However, changes in the abundance of several tricarboxylic acid cycle enzymes (e.g. aconitase, succinate dehydrogenase) suggest that tidally-acclimated mussels are also primed to sense reactive oxygen species (ROS) and limit their production, respectively. These findings are further supported by higher abundances of several aldehyde dehydrogenases and thioredoxin peroxidase, which function as scavengers of aldehydes and ROS, common products of lipid peroxidation. Finally, tidally-acclimated mussels are also more responsive to changes in cytoskeletal and vesicular trafficking dynamics in response to acute anoxia. Together, our analysis showed that proteostasis, energy metabolism, oxidative stress and cytoskeletal and trafficking processes are all involved in priming tidally-acclimated mussels to respond more dynamically to acute emersion-induced anoxia in Mytilus gill

Are Circadian cycles the dominant proteome rhythym in the intertidal mussel Mytilus californianus?

Mytilus californianus, also known as the California mussel, is a marine bivalve that is abundant along the West coast from Alaska to southern Baja California. They mainly reside in the upper-middle intertidal zone and cling to pier pilings and surf exposed rocks. They create multi-layered beds, which form a habitat for algae and many species of invertebrates. Intertidal mussels live in a naturally dynamic environment. It has previously been reported (Connor and Gracey, 2011) that the 24-hour circadian (day to night) rhythm of the intertidal mussel Mytilus californianus is primarily responsible for its rhythmic gene expression, as opposed to the 12.4-hour tidal cycles. Because tidal cycles challenge intertidal mussels through heat stress, salinity stress, hypoxia, and food availability, the dominance of the circadian cycle is surprising. However, transcriptomics may fail to detect up to half of the variation in the proteins that comprise the final functional phenotype of the organism. Using two-dimensional gel electrophoresis and mass spectrometry, we aimed to identify whether the proteome—the protein expression—of this organism also followed the same circadian rhythmic expression as its transcriptome

The proteomic response in the crustacean molting gland of land crab Gecarcinus lateralis in response to artificially induced molting throughout its molting cycle.

Molting in crustaceans is a highly complex physiological process involving negative regulation by two paired endocrine glands, the X-organ/sinus gland complex (XO/SG) and the Y-organ (YO). The XO/SG complex is responsible for making molt-inhibiting hormone (MIH) which negatively regulates synthesis of molting hormones (ecdysteroids) by the YO. Eyestalk ablation (ESA) removes the source of MIH and provides an experimental means to manipulate and induce molting, although the physiological effects of ESA on the YO have not been fully characterized. Analysis of gene expression in the XOs and YOs has lead to the development of a proposed molecular signaling pathway which regulates ecdysteroidogenesis and subsequent molting (ecdysis) in crustaceans. Results presented depict the changes in significantly different protein abundances in the YO over the course of the molting cycle (early, mid and late premolt) in crabs where 5 or more walking legs were lost, termed multiple leg autotomy (MLA). Proteins were characterized using two-dimensional gel electrophoresis and Delta2D software for statistical analysis. Future analysis will determine whether ESA can effectively mimic premolt conditions in the YO compared to the natural molting progression through protein identification by MALDI-TOF mass spectrometry. This will further resolve the metabolic and physiological changes associated with the transitions experienced by the YO throughout the molting stages. Determining the efficacy of ESA as a means to induce molting and determining molecular regulation of crustacean molting has broad economic impacts for crustacean fisheries as industry demands increase

The distribution of 4-nonylphenol in marine organisms of North American Pacific Coast estuaries

One of the chemical breakdown products of nonylphenol ethoxylates, 4-nonylphenol (4-NP), accumulates in organisms and is of concern as an environmental pollutant due to its endocrine disrupting effects. We measured 4-NP levels in the seawater, sediment, and twelve organisms within the California estuary, Morro Bay, and examined biomagnification of 4-NP using stable isotope abundances (δ15N and δ13C) to quantify trophic position. 4-NP concentrations in organisms from Morro Bay included 25000 ± 8600 ng g−1 lw in liver of California sea lion, 14000 ± 5600 ng g−1 lw in liver of harbor porpoise, 138000 ± 55000 ng g−1 lw in liver of sea otters, 15700 ± 3600 ng g−1 lw in liver of seabirds, 36100 ± 6100 ng g−1 lw in arrow goby fish, 62800 ± 28400 ng g−1 lw in oysters, and 12700 ± 1300 ng g−1 lw in mussels. 4-NP levels generally showed a pattern of trophic dilution among organisms in Morro Bay, with exceptions of biomagnification observed between three trophic links: mussel to sea otter (BMF 10.9), oyster to sea otter (BMF 2.2), and arrow goby to staghorn sculpin (BMF 2.7). Our examination of other west coastestuaries of USA and Canada revealed that mean 4-NP concentrations in gobies and mussels from Morro Bay were significantly higher than those from a more urbanized estuary, San Francisco Bay (goby: 11100 ± 3800 ng g−1 lw) and from a remote estuary, Bamfield Inlet, Canada (goby: 9000 ± 900 ng g−1 lw, mussel: 6100 ± 700 ng g−1 lw). Relative to other estuaries worldwide, 4-NP levels in seawater (0.42 ± 0.16 μg L−1) and sediment (53 ± 14 ng g−1 dw) of Morro Bay are low, but gobies and oysters have higher 4-NP levels than comparable fauna

Phytochrome A Regulates Carbon Flux in Dark Grown Tomato Seedlings

Phytochromes comprise a small family of photoreceptors with which plants gather environmental information that they use to make developmental decisions, from germination to photomorphogenesis to fruit development. Most phytochromes are activated by red light and de-activated by far-red light, but phytochrome A (phyA) is responsive to both and plays an important role during the well-studied transition of seedlings from dark to light growth. The role of phytochromes during skotomorphogenesis (dark development) prior to reaching light, however, has received considerably less attention although previous studies have suggested that phytochrome must play a role even in the dark. We profiled proteomic and transcriptomic seedling responses in tomato during the transition from dark to light growth and found that phyA participates in the regulation of carbon flux through major primary metabolic pathways, such as glycolysis, beta-oxidation, and the tricarboxylic acid (TCA) cycle. Additionally, phyA is involved in the attenuation of root growth soon after reaching light, possibly via control of sucrose allocation throughout the seedling by fine-tuning the expression levels of several sucrose transporters of the SWEET gene family even before the seedling reaches the light. Presumably, by participating in the control of major metabolic pathways, phyA sets the stage for photomorphogenesis for the dark grown seedling in anticipation of light

Ichthyosporidium weissii n. sp. (Microsporidia) Infecting the Arrow Goby (Clevelandia ios)

Gonadal infections by a novel microsporidium were discovered in 34% (13/38) of arrow gobies, Clevelandia ios, sampled over a 3-yr period from Morro Bay Marina in Morro Bay, California. Gonadal tumors had been reported in arrow gobies from this geographic area. The infected gonads, found primarily in females, typically appeared grossly as large, white-gray firm and lobulated masses. Histological examination revealed large, multilobate xenomas within the ovaries and no evidence of neoplasia. Typical of the genus Ichthyosporidium, the large xenomas were filled with developmental stages and pleomorphic spores. Wet mount preparations showed two general spore types: microspores with mean length of 6.2 (7.04.9, SD=0.6, N=20) mu m and mean width of 4.3 (5.32.9, SD=0.8) mu m; and less numerous macrospores with mean length of 8.5 (10.17.1, SD=1.0, N=10) mu m and mean width of 5.5 (6.24.8, SD=0.5) mu m. Transmission electron microscopy demonstrated stages consistent with the genus and 3550 turns of the polar filament. Small subunit rDNA gene sequence analysis revealed that the parasite from arrow gobies was most closely related to, but distinct from Ichthyosporidium sp. based on sequences available in GenBank. We conclude that this microsporidium represents a new species of Ichthyosporidium, the first species of this genus described from a member of the family Gobiidae and from the Pacific Ocean.This is the publisher’s final pdf. The published article is copyrighted by Wiley and can be found at: http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1550-7408Keywords: Parasite, Gonads, Phylogeny, Neoplasia, Electron microscopy, New specie

Pre- and early-postnatal nutrition modify gene and protein expressions of muscle energy metabolism markers and phospholipid fatty acid composition in a muscle type specific manner in sheep.

We previously reported that undernutrition in late fetal life reduced whole-body insulin sensitivity in adult sheep, irrespective of dietary exposure in early postnatal life. Skeletal muscle may play an important role in control of insulin action. We therefore studied a range of putative key muscle determinants of insulin signalling in two types of skeletal muscles (longissimus dorsi (LD) and biceps femoris (BF)) and in the cardiac muscle (ventriculus sinister cordis (VSC)) of sheep from the same experiment. Twin-bearing ewes were fed either 100% (NORM) or 50% (LOW) of their energy and protein requirements during the last trimester of gestation. From day-3 postpartum to 6-months of age (around puberty), twin offspring received a high-carbohydrate-high-fat (HCHF) or a moderate-conventional (CONV) diet, whereafter all males were slaughtered. Females were subsequently raised on a moderate diet and slaughtered at 2-years of age (young adults). The only long-term consequences of fetal undernutrition observed in adult offspring were lower expressions of the insulin responsive glucose transporter 4 (GLUT4) protein and peroxisome proliferator-activated receptor gamma, coactivator 1α (PGC1α) mRNA in BF, but increased PGC1α expression in VSC. Interestingly, the HCHF diet in early postnatal life was associated with somewhat paradoxically increased expressions in LD of a range of genes (but not proteins) related to glucose uptake, insulin signalling and fatty acid oxidation. Except for fatty acid oxidation genes, these changes persisted into adulthood. No persistent expression changes were observed in BF and VSC. The HCHF diet increased phospholipid ratios of n-6/n-3 polyunsaturated fatty acids in all muscles, even in adults fed identical diets for 1½ years. In conclusion, early postnatal, but not late gestation, nutrition had long-term consequences for a number of determinants of insulin action and metabolism in LD. Tissues other than muscle may account for reduced whole body insulin sensitivity in adult LOW sheep