Deep quantitative proteomics of North American Pacific coast star tunicate (Botryllus schlosseri).

Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress-induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep-coverage LCMS, revealing up to 4930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low-quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label-free quantitation of 3426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of single systems from a laboratory-raised with two field-collected populations revealed (1) a more unique proteome in the laboratory-raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were distinct in laboratory-cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate's deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research on B. schlosseri with proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions.

Exogenous iodide ameliorates perchlorate-induced thyroid phenotypes in threespine stickleback.

Perchlorate is a ubiquitous environmental contaminant that has widespread endocrine disrupting effects in vertebrates, including threespine stickleback (Gasterosteus aculeatus). The target of perchlorate is thyroid tissue where it induces changes in the organization, activation, and morphology of thyroid follicles and surrounding tissues. To test the hypothesis that some phenotypes of perchlorate toxicity are not mediated by thyroid hormone, we chronically exposed stickleback beginning at fertilization to perchlorate (10, 30, 100ppm) or control water with and without supplementation of either iodide or thyroxine (T4). Stickleback were sampled across a one-year timespan to identify potential differences in responses to treatment combinations before and after sexual maturation. We found that most thyroid histomorphological phenotypes induced by perchlorate (follicle proliferation, reduced follicle area (adults only), colloid depletion, thyrocyte hypertrophy (subadults only)) were significantly ameliorated by exogenous iodide supplementation. In contrast, treatment with exogenous T4 did not correct any of the thyroid-specific histopathologies induced by perchlorate. Whole-body thyroid hormone concentrations were not significantly affected by perchlorate exposure; however, supplementation with iodide and T4 significantly increased T4 concentrations. This study also revealed an increased erythrocyte area in the thyroid region of perchlorate-exposed adults, while lipid droplet number increased in perchlorate-exposed subadults. Increased erythrocyte area was ameliorated by both iodide and T4, while neither supplement was able to correct lipid droplet number. Our finding on lipid droplets indicates that exposure to perchlorate in early development may have obesogenic effects.

Perchlorate exposure does not modulate temporal variation of whole-body thyroid and androgen hormone content in threespine stickleback.

Previously we showed that exposure of threespine stickleback (Gasterosteus aculeatus) to the endocrine disruptor perchlorate results in pronounced structural changes in thyroid and gonad, while surprisingly, whole-body thyroid hormone concentrations remain unaffected. To test for hormone titer variations on a finer scale, we evaluated the interactive effects of time (diel and reproductive season) and perchlorate exposure on whole-body contents of triiodothyronine (T3), thyroxine (T4), and 11-ketotestosterone (11-KT) in captive stickleback. Adult stickleback were exposed to 100ppm perchlorate or control water and sampled at 4-h intervals across the 24-hday and at one time-point (1100h) weekly across the reproductive season (May-July). Neither whole-body T3 nor T4 concentration significantly differed across the day in control or perchlorate treated stickleback. Across the reproductive season, whole-body T3 concentration remained stable while T4 significantly increased. However, neither hormone concentration was significantly affected by perchlorate, verifying our previous studies. The concentration of whole-body 11-KT, a major fish androgen, displayed significant diel variation and also steadily declined across the reproductive season in untreated males; perchlorate exposure did not influence the concentration of 11-KT in either diel or reproductive season schedules. Diel and reproductive season variations in 11-KT content in male stickleback are likely related to reproductive physiology and behavior. The observed increase in T4 content across the reproductive season may be reflective of increased energy investment in reproduction near the end of the life cycle.

Salinity-induced regulation of the myo-inositol biosynthesis pathway in tilapia gill epithelium.

The myo-inositol biosynthesis (MIB) pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol that protects cells from osmotic stress. Using proteomics, the enzymes that constitute the MIB pathway, myo-inositol phosphate synthase (MIPS) and inositol monophosphatase 1 (IMPA1), are identified in tilapia (Oreochromis mossambicus) gill epithelium. Targeted, quantitative, label-free proteomics reveals that they are both upregulated during salinity stress. Upregulation is stronger when fish are exposed to severe (34 ppt acute and 90 ppt gradual) relative to moderate (70 ppt gradual) salinity stress. IMPA1 always responds more strongly than MIPS, suggesting that MIPS is more stable during salinity stress. MIPS is N-terminally acetylated and the corresponding peptide increases proportionally to MIPS protein, while non-acetylated N-terminal peptide is not detectable, indicating that MIPS acetylation is constitutive and may serve to stabilize the protein. Hyperosmotic induction of MIPS and IMPA1 is confirmed using western blot and real-time qPCR and is much higher at the mRNA than at the protein level. Two distinct MIPS mRNA variants are expressed in the gill, but one is more strongly regulated by salinity than the other. A single MIPS gene is encoded in the tilapia genome whereas the zebrafish genome lacks MIPS entirely. The genome of euryhaline tilapia contains four IMPA genes, two of which are expressed, but only one is salinity regulated in gill epithelium. The genome of stenohaline zebrafish contains a single IMPA gene. We conclude that the MIB pathway represents a major salinity stress coping mechanism that is regulated at multiple levels in euryhaline fish but absent in stenohaline zebrafish.

Tilapia (Oreochromis mossambicus) brain cells respond to hyperosmotic challenge by inducing myo-inositol biosynthesis.

This study aimed to determine the regulation of the de novo myo-inositol biosynthetic (MIB) pathway in Mozambique tilapia (Oreochromis mossambicus) brain following acute (25 ppt) and chronic (30, 60 and 90 ppt) salinity acclimations. The MIB pathway plays an important role in accumulating the compatible osmolyte, myo-inositol, in cells in response to hyperosmotic challenge and consists of two enzymes, myo-inositol phosphate synthase and inositol monophosphatase. In tilapia brain, MIB enzyme transcriptional regulation was found to robustly increase in a time (acute acclimation) or dose (chronic acclimation) dependent manner. Blood plasma osmolality and Na(+) and Cl(-) concentrations were also measured and significantly increased in response to both acute and chronic salinity challenges. Interestingly, highly significant positive correlations were found between MIB enzyme mRNA and blood plasma osmolality in both acute and chronic salinity acclimations. Additionally, a mass spectrometry assay was established and used to quantify total myo-inositol concentration in tilapia brain, which closely mirrored the hyperosmotic MIB pathway induction. Thus, myo-inositol is a major compatible osmolyte that is accumulated in brain cells when exposed to acute and chronic hyperosmotic challenge. These data show that the MIB pathway is highly induced in response to environmental salinity challenge in tilapia brain and that this induction is likely prompted by increases in blood plasma osmolality. Because the MIB pathway uses glucose-6-phosphate as a substrate and large amounts of myo-inositol are being synthesized, our data also illustrate that the MIB pathway likely contributes to the high energetic demand posed by salinity challenge.

Improved Media Formulations for Primary Cell Cultures Derived from a Colonial Urochordate.

The cultivation of marine invertebrate cells in vitro has garnered significant attention due to the availability of diverse cell types and cellular potentialities in comparison to vertebrates and particularly in response to the demand for a multitude of applications. While cells in the colonial urochordate Botryllus schlosseri have a very high potential for omnipotent differentiation, no proliferating cell line has been established in Botryllus, with results indicating that cell divisions cease 24-72 h post initiation. This research assessed how various Botryllus blood cell types respond to in vitro conditions by utilizing five different refinements of cell culture media (TGM1-TGM5). During the initial week of culture, there was a noticeable medium-dependent increase in the proliferation and viability of distinct blood cell types. Within less than one month from initiation, we developed medium-specific primary cultures, a discovery that supports larger efforts to develop cell type-specific cultures. Specific cell types were easily distinguished and classified based on their natural fluorescence properties using confocal microscopy. These results are in agreement with recent advances in marine invertebrate cell cultures, demonstrating the significance of optimized nutrient media for cell culture development and for cell selection.

Osmotic regulation and tissue localization of the myo-inositol biosynthesis pathway in tilapia (Oreochromis mossambicus) larvae.

The myo-inositol biosynthesis (MIB) pathway converts glucose-6-phosphate to the compatible osmolyte myo-inositol, which protects cells from salinity stress. We exposed tilapia larvae just after yolk sac resorption to various hypersaline environments and recorded robust induction of the enzymes that constitute the MIB pathway, myo-inositol-phosphate synthase (MIPS), and inositol monophosphatase 1 (IMPA1). Strong up-regulation of these enzymes is evident at both mRNA (quantitative real-time PCR) and protein (densitometric analysis of Western blots) levels. The highest level of induction of these enzymes occurs at the highest salinity that larvae were exposed to (90 ppt). Less severe salinity stress causes a proportionately reduced induction of the MIB pathway. Two distinct MIPS mRNA variants are present in tilapia larvae and both are induced at comparable levels for all the salinity challenges tested (34, 70, and 90 ppt). Immunohistochemical localization of IMPA1 protein in sagittal sections of salinity stressed and control larvae identified tissues that are particularly potent in inducing the MIB pathway. These tissues include the skin (epidermis), gills, eye (ciliary epithelium) and heart. In particular, the epidermis directly facing the external milieu showed a very strong induction of IMPA1 immunoreactivity. IMPA1 induction in response to salinity stress was not observed in other tissues suggesting that tilapia larvae may also utilize compatible organic osmolytes other than solely myo-inositol for osmoprotection. We conclude that the MIB pathway plays an important role in protecting multiple (but not all) tissues of tilapia larvae from hyperosmotic salinity stress.

Derivation and osmotolerance characterization of three immortalized tilapia (Oreochromis mossambicus) cell lines.

Fish cell cultures are becoming more widely used models for investigating molecular mechanisms of physiological response to environmental challenge. In this study, we derived two immortalized Mozambique tilapia (Oreochromis mossambicus) cell lines from brain (OmB) and lip epithelium (OmL), and compared them to a previously immortalized bulbus arteriosus (TmB) cell line. The OmB and OmL cell lines were generated without or with Rho-associated kinase (ROCK) inhibitor/3T3 feeder layer supplementation. Although both approaches were successful, ROCK inhibitor/feeder layer supplementation was found to offer the advantages of selecting for epithelial-like cell type and decreasing time to immortalization. After immortalization (≥ passage 5), we characterized the proteomes of the newly derived cell lines (OmB and OmL) using LCMS and identified several unique cell markers for each line. Subsequently, osmotolerance for each of the three cell lines following acute exposure to elevated sodium chloride was evaluated. The acute maximum osmotolerance of these tilapia cell lines (>700 mOsm/kg) was markedly higher than that of any other known vertebrate cell line, but was significantly higher in the epithelial-like OmL cell line. To validate the physiological relevance of these tilapia cell lines, we quantified the effects of acute hyperosmotic challenge (450 mOsm/kg and 700 mOsm/kg) on the transcriptional regulation of two enzymes involved in biosynthesis of the compatible organic osmolyte, myo-inositol. Both enzymes were found to be robustly upregulated in all three tilapia cell lines. Therefore, the newly established tilapia cells lines represent valuable tools for studying molecular mechanisms involved in the osmotic stress response of euryhaline fish.