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Mar. Drugs 2014, 12(6), 3770-3791; doi:10.3390/md12063770
Abstract: Many bioactive products from benthic invertebrates mediating ecological interactions have proved to reduce predation, but their mechanisms of action, and their molecular identities, are usually unknown. It was suggested, yet scarcely investigated, that nutritional quality interferes with defensive metabolites. This means that antifeedants would be less effective when combined with energetically rich prey, and that higher amounts of defensive compounds would be needed for predator avoidance. We evaluated the effects of five types of repellents obtained from Antarctic invertebrates, in combination with diets of different energetic values. The compounds came from soft corals, ascidians and hexactinellid sponges; they included wax esters, alkaloids, a meroterpenoid, a steroid, and the recently described organic acid, glassponsine. Feeding repellency was tested through preference assays by preparing diets (alginate pearls) combining different energetic content and inorganic material. Experimental diets contained various concentrations of each repellent product, and were offered along with control compound-free pearls, to the Antarctic omnivore amphipod Cheirimedon femoratus. Meridianin alkaloids were the most active repellents, and wax esters were the least active when combined with foods of distinct energetic content. Our data show that levels of repellency vary for each compound, and that they perform differently when mixed with distinct assay foods. The natural products that interacted the most with energetic content were those occurring in nature at higher concentrations. The bioactivity of the remaining metabolites tested was found to depend on a threshold concentration, enough to elicit feeding repellence, independently from nutritional quality.
1. Introduction
One main topic in the field of marine chemical ecology is chemical defense, especially the investigation of secondary metabolites that provide protection from predation in potential prey. A series of questions arise when studying feeding repellents: how do they affect predators, are they toxic, or do they taste bad? Do chemicals require characteristic moieties to interact with specific receptors to be active as defenses? Are certain types of molecules more effective than others? What range of predators do metabolites affect? Are repellents energetically costly? May a secondary metabolite have no function? Do other metabolites (e.g., co-occurring bioactive secondary or primary metabolites, nutrients) interact with feeding repellents altering (enhancing or reducing) their effects? [1,2,3]. Some of these questions were approached in the present study under laboratory controlled conditions, using different isolated forms (pure compounds or mixtures of related metabolites) of chemical defenses, and assessing their levels of bioactivity and interaction with nutrients.
Predation is a dominant force in controlling populations of marine invertebrates, and prey organisms have evolved protective strategies, ranging from behavioral (nocturnal activity, rapid escape) and physical (spines, armor), to chemical mechanisms [4]. It is believed (but not fully proved) that palatability/distastefulness, and not toxicity, is responsible for the action of most defensive repellents, which elicit immediate aversive responses in potential predators [5,6,7]. The assumption of a metabolic cost for possessing repellents is ambiguous. Very few studies have examined the metabolic investments of defense in relation to an organism's energy budget (for maintenance, growth, reproduction). Energy may be saved if defenses are active in small concentrations, do not require detoxification for storage, or if they originate from dietary or symbiotic producers. To optimize costs, chemical defenses could be differentially allocated in parts that are more susceptible to attacks, or be produced only when needed, as predicted by the optimal defense theory (ODT) [8]. There are also secondary metabolites that accumulate as by-products of the synthesis of other compounds, and have no known specific function [9]. Indeed, from the enormous diversity of chemicals from benthic invertebrates, ecological function has only been established in a tiny fraction of them [2,6,10,11].
The activity of antipredatory compounds might be altered by the presence of other compounds or physical devices, sometimes acting in an additive or synergistic manner. However, evaluating such effects is of great complexity, as is illustrated in an exchange of controversial publications [12,13,14]. Dietary components, like attractants that enhance feeding (i.e., amino acids, nutrients), may instead reduce repellent activity, interacting in an antagonistic way. The nutritional quality of potential prey is therefore relevant, since it is likely that the same sensory processes that predators use to reject defensive metabolites are also used to perceive nutrients. Thus, prey with low nutritional quality may be rejected at lower levels of chemical defense, and conversely, natural products might only be deterrent at high concentrations in more nutritious items. Experimentally, high energy assay foods may mask minimally effective defensive metabolites, and consequently, products with weak bioactive properties may only cause deterrence along with low energy foods [15,16].
Furthermore, the relationships between chemical structure and defensive activity have not yet been addressed. Compounds of several classes and different polarities are described among feeding deterrents, from non-polar terpenoids to polar glycosides, some with broad effectiveness against a wide range of predators [2,6,10,17,18,19]. This suggests that chemoreceptive responses of diverse predatory taxa are similar at the molecular level [7]. There are likewise metabolites displaying multiple defensive roles, for example antipredatory and antifouling [20,21], representing energy saving defenses against different potential enemies, according to the Optimality Theory (OT) [22].
For all intents and purposes, most of the questions listed above remain unsolved, and, so far, little information is available. Besides, the research effort to understand antipredatory defensive strategies is notably biased, since it has been much more intensive in zones like the Caribbean than in other marine regions, in particular the Poles [1,2,3,6]. Research on the Southern Ocean regarding ecological interactions mediated by chemistry has grown in the past years, and many natural products have been discovered, some with allelochemical functions (e.g., for reviews see [10,11,17,18,23]). The present study attempts to elucidate some of the obscure points yet to be understood, on how feeding repellents operate in the presence of distinct relative amounts of nutrients, focusing on a benthic predator–prey scenario in a remote geographic area, Antarctica. In these polar ecosystems, predation (driven by invertebrate consumers, such as sea stars, nemertine worms and dense amphipod populations) is a selective force structuring benthic communities [24]. Following our previous investigations on defensive metabolites from Antarctic invertebrates with feeding repellent properties, the aims of this work are: (1) to evaluate how the antipredatory activity of selected natural products is affected by the energetic content of food (usually expressed in units of energy per mass; e.g., kJ·g−1); (2) to compare the potency/efficacy of the compounds tested as defenses, according to their chemical structure; and (3) to determine whether the efficiency of defensive metabolites (compound concentration required to yield bioactivity) has any relationship with the concentrations in which they appear in nature, and/or interference with the nutritional value of assay food items designed in order to mimic possible prey. The target compounds included alkaloids (meridianins A–G) from Aplidium ascidians [21,25], a steroid (5α(H)-cholestan-3-one) from hexactinellid sponges [26], wax esters from Alcyonium soft corals [27], a meroterpenoid (Rossinone B) from the ascidian A. fuegiense [21,28], and a new organic acid, glassponsine, from the glass sponge Anoxycalyx (Scolymastra) joubini [29]. These products were included in artificial diets of different energetic values, at distinct concentrations, and were then presented to a sympatric Antarctic amphipod predator in feeding experiments. Due to its ecological relevance as generalist consumer, we chose the lyssianasid Cheirimedon femoratus as the experimental predator for our assays.
2. Results and Discussion
Sufficient material (≈11 mg) of each of the isolated target compounds (10–12), and of the target mixtures of chemically related products (3–9) and (1–2), was successfully obtained for the experiments. Both mixtures are composed of metabolites of the same family type: Within the wax fraction, both wax esters 1 and 2 appear in approximately the same proportion ~1:1 [27]; whereas the meridianin (A–G) alkaloid mix (3–9) contains a major component constituted by meridianins A–E (3–7) and a minor one corresponding to F–G meridianins (8–9), in a relative proportion of ~94:6. These proportions may exhibit slight variabilities among colonies. Nevertheless, the isolated forms of A–G meridianins have demonstrated in the past similar efficiency as feeding repellents [25]. The natural relative concentrations of the metabolites within both mixtures (1–2 and 3–9) were maintained (not modified after fractionation) in the bioassays here performed. The natural products 1–11 had been previously isolated from our Antarctic sample collections, all of them demonstrating feeding repellent properties towards relevant sympatric predators: the sea star Odontaster validus and the amphipod Cheirimedon femoratus [21,25,26,27]. In preliminary experiments, the newly described glassponsine (12) did not cause rejection in feeding tests at its natural concentration (~2.34 mg·g−1; [29] and present study), but repellent activity was confirmed at higher concentrations (see below). Specific data of the five target compound types can be found in Table 1, while the information on the setup design of the experiments is illustrated in Figure 1.
As a consequence of exhaustive analyses, glassponsine (12) was recently recovered as a new compound from several individuals of the common Antarctic Hexactinellid sponge Anoxycalyx (Scolymastra) joubini [29], and was here assessed as a feeding deterrent product for the first time. Conspecific samples from collections coming from different geographic areas have never afforded molecules similar to 12 [10,26]. Glassponsine 12 was recovered as an abundant sulfonate salt (see Table 1). It has a close structural relationship with the widely distributed taurine, responsible for many biological roles in animals (i.e., osmolytes in deep-sea organisms) and a major constituent of bile [30]. Related sulfonate acids, some found in demosponges and also common in bacterial membranes, are important antibiotics in the production of sulfa drugs [10,11,31]. The scant organic content and high spicule concentration of glass sponges make them supposedly already discouraging to predators, and needless to produce secondary metabolites [32]. Frequently though, marine organisms reveal variability in chemical profiles and in defensive activities within the species or even population level, and this is often attributed to symbiotic producers [33,34,35,36]. Bacterial symbionts, due to the extremely reduced mesohyl of Hexactinellida, are believed to be unsubstantial in this class of Porifera [32] when compared with Demospongiae [37]. Glassponsine might originate from microbial associates, or either be produced by host cells of a distinctive population of glass sponges adapted to particular conditions, and from which our specimens were recovered. In any case, from an ecological evolutionary perspective, intraspecific variabilities in bioactivity patterns may promote segregation, favoring the selection for certain defended phenotypes. These issues still require further investigation.
Analysis with Thin Layer Chromatography (TLC) were used to evaluate chemical changes in the alginate pearls during experimentation; these showed that the metabolites added to the artificial feeding pearls mixtures always remained unchanged after the bioassays.
|Compounds' Name and Chemical Structure
|Chemical type
|Wax ester
|[N]Comp in DWTOTAL
|25 mg·g−1
|Organic fraction
|Ether (EE)
|Source organism
|Soft coral
|Species name
|Alcyonium haddoni
|Sample location
|Deception Island
|Chemical type
|Indole alkaloid
|[N]Comp DWTOTAL
|24.3 mg·g−1
|Organic fraction
|Ether (EE)
|Source organism
|Colonial ascidian
|Species name
|Aplidium falklandicum
|Sample location
|Weddell Sea
|Chemical type
|[N]Comp DWTOTAL
|5.1 mg·g−1 *
|Organic fraction
|Ether (EE)
|Source organism
|Colonial ascidian
|Species name
|Aplidium fuegiense
|Sample location
|Weddell Sea
|Chemical type
|[N]Comp DWTOTAL
|2.8 mg·g−1
|Organic fraction
|Ether (EE)
|Source organism
|Glass sponge
|Species name
|Rossella nuda
|Sample location
|Weddell Sea
|Chemical type
|Sulfonate acid
|[N]Comp DWTOTAL
|2.34 mg·g−1
|Organic fraction
|Butanol (BE)
|Source organism
|Glass sponge
|Species name
|Anoxycalyx (Scolimastra) joubini
|Sample location
|Weddell Sea
[N]Comp values refer to average yields obtained from this study and previous publications [21,25,26,27]; * Natural concentrations of rossinone (10) come from inner regions of the colonies after dissection.
2.1. Control (Compound-Free) Diets
The three prepared alginate-based food pearls, containing different proportions of feeding stimulant (Phytoplan®) and sand, produced significantly different ingestion rates in the amphipod Cheirimedon femoratus (Figure 2) according to the one-way ANOVA analysis (F(3,2) = 38.107, p < 0.001 **). The energetically intermediate diet (100 mg feeding stimulant, 100 mg sand) was the most consumed (p < 0.001 ** Tukey (HDS) post hoc tests, when compared with the other two diets). The least nutritive food pearls (50 mg feeding stimulant, 150 mg sand), were more intensively eaten than those with 200 mg Phytoplan® and no sand added (p < 0.001 **), thus ranking ingestion rates per diet are: 100 > 50 > 200 mg Phytoplan®. When testing preferences between the three diets, the energetically richest pearls (200 mg Phytoplan®) were significantly preferred compared with the other two feeding pearls (p < 0.05 * in both comparisons according to Wilcoxon Exact tests). Pearls prepared with 100 mg food source tended to be preferred compared with those of 50 mg Phytoplan® and 150 mg sand, yet the difference was not significant (p = 0.089). These preferences may be resumed as: 200 > 100 ≥ 50 mg Phytoplan® (see Table 2; Figure 2). Consequently, amphipods prefer the energetically richest (200 mg Phytoplan®) diet, but require lesser amounts to reach saturation compared to the lower energy diets.
|Diets Contrasted
|Feeding Preferences
|Ingestion Rates
|PhytoPlan® Content
|Preference Result
|Wilcoxon Test
|Ingestion Result
|Tukey (HDS)
|200 mg vs. 100 mg
|200 mg > 100 mg
|p = 0.036 *
|200 mg < 100 mg
|p < 0.001 **
|200 mg vs. 50 mg
|200 mg > 50 mg
|p = 0.014 *
|200 mg < 50 mg
|p < 0.001 **
|100 mg vs. 50 mg
|100 mg > 50 mg
|p = 0.089 n.s.
|100 mg > 50 mg
|p < 0.001 **
Feeding preferences of the amphipod Cheirimedon femoratus were towards the energetically richest non-mineralized diets, in comparison with poorer foods containing inorganic material reflected palatability (feeding attractiveness); whereas the pattern of ingestion, with highest consumption of energetically intermediate foods, and lowest with most nutritive ones, is likely related to satiation and digestibility. While nutritious items are probably more attractive, their high energetic values make them also more satiating, leading to a lower consumption when compared with less nutritive diets. Alternatively, energetically poor food items, with high inorganic content, may cause lower ingestion rates than what would be expected, due to an excess of indigestible inert load, making their digestion too costly, less profitable, and therefore not worthwhile for consumers. Compensatory feeding is a well-described phenomenon by which animals compensate for ingesting energetically poor diets by increasing the rate of consumption. Indigestible components (sand) in low nutrient density foods might, however, offset compensatory ingestion, since an enhanced intake also entails accumulation of profitless substances [38,39]. Simultaneously, according to the digestive rate model (DRM), predators select for most nutritive, easily digestible items [7,40,41]. Our findings are in agreement with those predictions; food with intermediate energetic and inorganic content provided the highest ingestion rates and intermediate feeding preferences.
2.2. Feeding Repellent Activities of Target Bioactive Metabolites (1–12)
Activities of the five different metabolite types towards the amphipod Cheirimedon femoratus differed depending on concentration and diet energy content (Table 3; Supplementary Figure S1). The meridianin A–G (3–9) mixture was the most active (Wilcoxon Exact Tests; p < 0.05 *), causing repellency to the amphipod in seven of the nine assays performed (77.8% active tests). No deterrence was observed, only in tests with the intermediate and lowest compound concentrations (5, 2.5 mg·g−1) and added to the richest diet (pearls made with 200 mg Phytoplan® and no sand). Glassponsine (12) with 33.3%, active results, and rossinone B (10) with 44.4% were repellent at their highest concentrations (10 mg·g−1) in all three types of artificial foods. Rossinone B (10) was also repellent when incorporated to pearls of highest energetic values (200 mg Phytoplan®) at intermediate concentration (5 mg·g−1). The steroid 5α(H)-cholestan-3-one (11) yielded 11.1% incidence of activity, only eliciting repellence at the highest compound concentration (10 mg·g−1) included in the energetically richest diet type (200 mg Phytoplan®). Finally, the wax esters (1–2) were similarly active only in one test (11.1% activty), but only when incorporated at highest experimental concentration (10 mg·g−1) within pearls of lowest nutritional content (50 mg Phytoplan® and 150 mg sand). These results are summarized in Table 3 and Supplementary Figure S1.
|PhytoPlan (mg)
|Wax esters (1–2)
|Meridianins A–G (3–9)
|Rossinone B (10)
|5α(H)-cholestan-3-one (11)
|Glassponsine (12)
* Color codes corresponding to percentage intervals of relative average differences of ingestion rates between control vs. treatment food pearls for the 15 replicate tests of each experiment.
2.3. Interference between Energetic Content and Compounds (1–12) Deterrent Bioactivity
According to G-Tests of independence calculated on 3 × 3 contingency tables constructed with the variables "Assay diet" and "Compound experimental concentration", there were different levels of interaction between the artificial feeding pearls' energetic content and compound efficacy as repellents for the five compound types assessed (1–12). Meridianins 3–9 exhibited the highest grade of interference in their feeding repellent properties (G = 13.5546; p < 0.01 **, Figure 3), experiencing a reduction in the efficiency (e.g., requirement of higher compound concentrations to provide deterrence) when the energetic content of assay diets increased. The next product whereby bioactivity was offset by energy content was the wax esters 1–2 (G = 9.493; p < 0.05 *). For the rest of the metabolites analyzed, Rossinone 10, Cholestan 11, Glassponsine 12, no statistic significance (G = 8.626, 6.037, 1.884 respectively; p > 0.05 n.s.) supported the existence of dependence/interaction between the experimental diets and bioactive compound concentrations; e.g., reduced efficacy of defensive metabolites as antifeedants in higher energy assay food items (Figure 3).
* The larger the likelihood ratio statistic G is, the larger is the grade of interaction between variables (i.e., "Assay diets" vs. "Compound testing concentrations"), and the lower is the p-value.
Glassponsine (12) showed no feeding repellent activity at 2.5 mg·g−1, close to its natural concentration (2.34 mg·g−1), but highest assay concentrations (10 mg·g−1) did elicit rejection by Cheirimedon femoratus. The ketosteroid 5α(H)-cholestan-3-one (11) is another compound from Antarctic glass sponges, in this case Rossella nuda, and it appears in variable amounts in several species of hexactinellids. This metabolite was reported as an effective repellent at a mean concentration of 2.5 mg·g−1, yet its defensive role is considered to be minor and likely supported by synergism with other co-occurring agents [26]. It shows unpredictable levels of activity, since no correlation is found between concentration and feeding rejection in the sponge extracts containing the steroid [26]. 5α(H)-cholestan-3-one, like other steroid ketones, is a by-product of the cholesterol degradation route, and its accumulation is a matter of debate, as either a metabolic dead end or as a discrete functional defense [9,42]. In the present study, only one experiment yielded significant unpalatability at the highest compound concentration.
Wax esters (1–2) were purified from Alcyonium haddoni, but they are frequent in Antarctic Alcyonium soft corals in general [27], as well as in other marine organisms. Wax esters are the main energy resources in anthozoans (corals), and are significantly dependent on the metabolic demands of the colony, which explains their highly variable tissue concentrations [43]. They are indigestible for most consumers, and possess additional unpalatable activity towards relevant Antarctic predators at diverse tissue concentrations, starting at ~5 mg·g−1 for C. femoratus amphipods, according to previous studies [27]. In the experiments here performed, however, only in one case did high wax concentration (10 mg·g−1) provoke amphipods to reject feeding pearls (Table 1 and Table 3; Supplementary Figure S1). Wax esters 1–2 play a major primary metabolic function as lipidic reserves, while providing deterrent properties; this dual role turns them into efficacious energy-saving defensive metabolites. Similarly to the aforementioned ketosteroid 11, waxes are thought to cooperate in an additive way with other more potent bioactive secondary metabolites. In corals, they presumably interact with terpenoids (e.g., illudalane alcyopterosins in Antarctic Alcyonium), to provide a more effective protection [27].
The remaining products consisted of potent chemical defenses coming from ascidians and of secondary metabolism origin. Rossinone B (10) was first described from an Antarctic colonial Aplidium sp. [44], and was subsequently isolated from Aplidium fuegiense [28]. It is a meroterpenoid preferentially stored towards the internal parts of the colonies, and with strong feeding repellent properties at inner natural concentrations (5.1 mg·g−1) [21,28]. In the present study, rossinone B displayed activity with the highest and intermediate concentrations (5 and 10 mg·g−1), but not in all cases for the intermediate amount. Finally, meridianins A–G (3–9) are indole alkaloids originally reported from Aplidium meridianum [45], and later discovered in Aplidium falklandicum [25]. Meridianins are very potent protein kinase inhibitors of pharmacological interest [11,46]. Ecologically, they have multiple defensive roles that extend beyond antipredatory to antifouling, presumably turning them into multipurpose repellents [22]; however, their high tissue amount puts into question the energy-saving theory, unless they are produced by symbionts. In nature, meridianins occur as a complex mixture of related alkaloids, separately exhibiting some types of bioactivity at concentrations as low as 0.75 mg·g−1 [25]. Several other repellents that are effective as isolated forms, (e.g., tambjamines, water-borne steroids) appear normally as families of related minor metabolites, and have enhanced effects in combination. Apparently, producing mixtures of similar active chemicals adds more signals to the bioactive constituent [25,47,48]. Meridianins A–G occurred in abundant quantities here, up to 24.3 mg·g−1 distributed throughout the whole colony (in accordance to previous studies [21,25]), yet frequently they may be slightly more concentrated towards outer regions. They are impressively powerful feeding repellents, even at substantially lower concentrations than those calculated as natural [21,25]. In this study, the meridianin mixture was the most effective repellent causing amphipod rejection at all testing concentrations (see Table 1 and Table 3; and Supplementary Figure S1).
The evolution of chemical defenses and the responses exhibited by consumers cannot be understood in isolation from dietary ingestion. Similarly, nutrition must integrate the effects of non-nutrient components, including indigestible substances and secondary metabolites. One mode of antipredatory defensive strategy is proposed to use this antagonistic interaction between nutrients and repellents. Thus, prey may become able to reduce palatability by combining low nutrient content with the presence of distasteful secondary metabolites. Following these assumptions, chemical defenses should be more (or only) effective when incorporated into nutritionally low-quality diets. However, there is still scant empirical evidence, with only a few compounds tested, to support these arguments [7,12,16]. Our experimental data show that levels of repellent activity vary for each compound type (1–12), and that they perform differently when mixed with nutritionally distinct assay foods. The products that interacted the most with energetic content were those that occur in nature at higher tissue concentrations, according to G-Tests of independence (Figure 3). Those were the wax esters (1–2) and meridianins A–G (3–9). More energetic diets required higher quantities of these metabolites to produce repellent effects; efficient bioactivity, however, was acquired with meridianins at much lower concentrations when compared with wax esters. Accordingly, waxes were only active at maximum concentrations in the poorest diets; whereas meridianins always elicited repellency, except with the most nutritious diets, for which maximum concentration of the mix was needed to provide effective amphipod rejection. Instead, rossinone B (10), 5α(H)-cholestan-3-one (11) and glassponsine (12), which appear in much lower quantities (five- to ten-fold lower) within the source organisms, behaved independently with respect to food quality (see Table 3 and Figure 3). For these compounds maximum concentrations were active irrespective of the diet richness, except for the ketosteroid (11), which was surprisingly only active in most nutritious pearls. Rossinone B (10), moreover, elicited feeding rejection at medium concentrations within richest assay foods.
The most potent feeding repellents were by far the meridianins A–G, followed by the rossinone B, and then glassponsine; 5α(H)-cholestan-3-one and wax esters presented lower activities. According to what is routinely assumed about antipredatory chemistry, secondary metabolites (alkaloids 3–9, meroterpene 10 and sulfonate acid 12) demonstrated significantly higher levels of defense compared with compounds deriving from primary metabolic routes (wax esters 1–2 and steroid 11). Moreover, the fact that meridianin fractions were found as the most potent feeding repellents at any concentration, agrees with traditional theories, in that nitrogen-containing metabolites (such as alkaloids) should be bioactive at much lower concentrations than non-nitrogenous defenses such as phenolics or terpenes [1]. Some inorganic components (e.g., spicles, sclerites, calcified plant structures) have been attributed a defensive role, as to be able to act in a synergistic way with repellent metabolites against predators [12]. This synergism was not intended to be analyzed here, as sand was added to the prepared foods only to maintain a constant dry weight while changing the energetic content. No enhanced bioactivities were observed either way with pearls containing sand compared with sand-free ones, in combination with repellents. Remarkably, calculations of natural concentrations here and in most published studies, consider portions of animals that might not be susceptible to a cellular or glandular specific allocation of defensive metabolites, being thus quite conservative. Therefore, at smaller scales, these compounds could be even more concentrated, providing stronger effects. This was not a main topic of the present study, but it is important to consider an antipredatory activity of products at higher concentrations than those calculated as natural. In fact, according to the optimal defense theory (ODT), an optimal anatomical storage of defenses is important also to avoid possible autotoxicity [3,8].
3. Experimental Section
3.1. Sample Collection and Extraction
Selected Antarctic invertebrate samples from the Eastern Weddell Sea, collected by trawling between 228 and 309 m depth during the ANT XXI/2 cruise (November 2003–January 2004) on board the R/V Polarstern (AWI—Alfred Wegener Institute, Bremerhaven, Germany), were recovered for the isolation of the target metabolites. These collections included colonial ascidian samples of the species Aplidium falklandicum Millar, 1960 and Aplidium fuegiense Cunningham, 1871; and hexactinellid sponge samples, Anoxycalyx (Scolymastra) joubini Topsent, 1916 and Rossella nuda Topsent, 1901. Moreover, colonies of the soft coral Alcyonium haddoni Wright and Studer, 1889 were collected at 15 m depth by scuba diving around Deception Island (South Shetland Archipelago, Antarctica) during the ACTIQUIM-2 campaign (December 2009–January 2010). Each single sample comprised several colonial clumps or individuals of each species from a collection site, which were grouped together for further experimentation and analysis. Pictures of fresh animals were taken on board, and a voucher portion of each sample was conserved in 10% formalin for taxonomy. The material was frozen at −20 °C, and sent to the University of Barcelona, where all five samples were classified to species level (Table 1).
Each of the five samples, consisting of various colonies/specimens, was thawed, cut in small pieces and extracted with acetone at room temperature while grinding the tissue with a mortar and pestle. In the particular case of Aplidium fueginese colonies were previously dissected into inner and outer body regions, and only the inner region (zooids), reported to store the target metabolite rossinone (10) [21,28], was processed. After removal of the solvent in vacuo, residual water was partitioned three times with diethyl ether (Et2O) and once with n-butanol. The organic phases were combined to obtain an ether fraction (EE) and a n-butanol fraction (BE). The respective organic solvents were evaporated under reduced pressure, providing dry EE and BE fractions and an aqueous residue. The dry crude fractions were weighed for calculations of yield. The weight of the material of the residual aqueous fraction was not included in the dry weight calculation. Four ether and one n-butanol fractions, each one obtained from one of the five samples of the analysis, were used for the chemical purifications of the five target metabolites 1–12 (see Section 3.3). Tissue concentrations for the isolated compounds, hereafter referred to as "natural concentrations," were calculated with respect to a sample's total dry weight: DWTOTAL = DW dry weight of the solid extracted remains + EE dry weight + BE dry weight (Table 1). We chose dry weight for tissue concentration calculations because it avoids issues of high variability of weight parameters related to water content in aquatic, soft-bodied samples.
3.2. Molecular Characterization
1H and 13C NMR spectra of samples dissolved in CDCl3 were recorded on DRX 600, Avance 400, and DPX 300 MHz Bruker spectrometers (Bruker BioSpin AG, Fällanden, Switzerland), with chemical shifts reported in ppm relative to CHCl3 (δ 7.26 for proton and δ 77.0 for carbon). Electro Spray Ionization Mass Spectroscopy (ESIMS) and High Resolution Electron Spray Ionization Mass Spectroscopy (HRESIMS) were measured on a Micromass Q-TOF Micro mass spectrometer coupled with a Waters Alliance 2695 HPLC (Waters S.A.S., En Yvelines Cedex, France). The instrument was calibrated with a polyethylene glycol (PEG) mixture representing molecular weights ranging from 200 to 1000 g/mol. Silica gel chromatography was performed using precoated Merck F254 plates and Merck Kieselgel 60 powder (Merck Millipore, Vimodrone (MI), Italy). HPLC purification was carried out on a Shimadzu LC-10 AD liquid chromatograph equipped with a UV SPD-10A wave-length detector (Shimadzu Europa GmbH, Duisburg, Germany).
3.3. Purification of Target Metabolites
Crude organic extracts (diethyl ether and butanol) obtained from our five invertebrate samples were transferred to ICB-CNR (Pozzuoli, Napoli, Italy), where they were further processed. They were screened by TLC using light petroleum ether/Et2O in different ratios (1:0, 8:2, 1:1, 2:8, 0:1), and chloroform/methanol 8:2. The plates were developed with CeSO4 revealing diverse spots, depending on each particular sample. Organic extracts containing the selected metabolites of interest for the study were fractionated through chromatography on Sephadex LH-20 (Sigma-Aldrich, Milano, Italy) with a 1:1 mixture of chloroform/methanol, and/or were submitted to silica gel (Merk Kiesegel 60, 0.063–0.2, Merck Millipore, Vimodrone (MI), Italy) column purification using light petroleum ether/Et2O/chloroform/methanol as eluents in different combinations and ratios, according to the nature of the target compounds. The following five metabolites were used for the bioassays (see Table 1):
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Wax esters (1–2): The lipophilic (Et2O) fraction from the soft coral Alcyonium haddoni was submitted to silica gel column purification to give a wax ester subfraction (Rf 0.9, light petroleum ether/Et2O, 9:1). LC-MS analysis (250 × 4.60 mm, Phenomenex Kromasil C18, 60 min gradient from 30% to 100% CH3OH in H2O) showed that the wax ester mixture comprised the two main components: wax (1) C34:1 (m/z 529 [M + Na]+), and wax (2) C32:1 (m/z 501 [M + Na]+) (both composed of a C18:1 monounsaturated fatty acid, and C16:0 and C14:0 saturated alcohol esters, respectively). 1–2 were assayed as a mixture, because they are the main components of the wax ester fraction in Alcyonium soft corals. For further details on purification and molecular determination of products 1 and 2 see [27];
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Meridianins A–G (3–9): Column chromatography of the ether fraction of the colonial ascidian Aplidium falklandicum, followed by a TLC purification with preparative (SiO2) plates (Merk Kiesegel 60 F254 0.50 and 1.00 mm), provided an abundant yellowish subfraction (Rf 0.63, chloroform/methanol, 8:2), which corresponded to the alkaloid mixture of meridianins A–G. These products (3–9) were tested as a mixture since in nature they occur together as a rich fraction of bioactively related derivatives. For details on purification and molecular determination of meridianins A–G see [21,25];
- -
Rossinone B (10): The Et2O inner extract from the ascidian Aplidium fuegiense was fractioned through column and TLC purifications, allowing to recover significant quantities of the meroterpenoid rossinone B (10) (Rf 0.65, light petroleum ether/Et2O, 2:8). Details on purification and molecular determination of product 10 may be found in [21,28];
- -
5α(H)-cholestan-3-one (11): The ether extract of the glass sponge Rossella nuda was fractioned by silica gel chromatography, using a gradient of light petroleum ether/Et2O. The fraction eluted with 10% of diethyl ether (Rf 0.51, light petroleum ether/Et2O, 8:2) contained pure 5α(H)-cholestan-3-one (11). More information on the isolation of product 11 is published in [26];
- -
Glassponsine (12): The fractionation of the BE fraction of the hexactinellid Anoxycalyx (Scolimastra) joubini on Si gel column chromatography (gradient 0%–100% metanol in chloroform) yielded a fraction containing a UV sensitive spot at Rf 0.15 (chloroform/methanol, 8:2). This fraction was further purified by preparative TLC chromatography (SiO2, chloroform/methanol, 65:35) to yield a recently reported pure compound, glassponsine 12, with a structure elucidated by spectroscopic methods elsewhere [29].
3.4. Artificial Food Preparation
Alginate caviar-textured feeding pearls were used in the assays. Gelling agents add little nutritional value, and the resulting artificially prepared diets allow homogeneous inclusion of ingredients, while controlling the nutritional content (determined by the quantity of food supplement added), and the concentration of metabolites under investigation. An added advantage of using alginate is that, unlike other gelling agents, it does not require heating, reducing chemical degradation. Phytoplan®, a spray-dried blend of several strains of phytoplankton (used for aquarium filter feeding invertebrates) was in our case the feeding source (30.3% protein, 13.6% carbohydrate and 13% of lipids, based on nutrition facts). Invertebrate tissues contain nutritionally valuable (e.g., protein) and inert components (e.g., mineralized skeletal inclusions: glass and/or calcareous spicules of sponges, and calcitic sclerites of corals and ascidians). Assay foods should include an equivalent mass of nutritionally inert matrix to mimic the mineralized inorganic skeletal elements naturally present in the groups studied. This was substituted by fine washed beach sand (0.0625 mm particule-size). Artificial diets were made with Kit Sferificacion® [49] similarly as in previous studies by our group [19,21,26,27]. Food pearls were prepared by mixing 5 mg·mL−1 of alginate (Algin® of Kit Sferificacion®) aqueous solution, along with three different quantities/concentrations of Phytoplan® (200, 100 and 50 mg; 66.7, 33.3 and 17 mg·mL−1), a compensating quantity of sand (0%, 50% and 75% of food's total dry mass) to maintain constant dry weight in the prepared artificial food mixes, and a drop of green or red food dye (Table 4; see next paragraph). This provided the production of three diets with constant dry weight (≈200 mg) but different nutritional quality, estimated in energetic values as ≈19, 9.5 and 4.75 KJ·g−1 in dry weight, respectively (according to the Atwater factor system [50]). Published analyses from several authors report energetic values of 5–6 KJ·g−1 dry wt in Antarctic hexactinellid sponges, which have a high spicule content and low organic matter and represent poorly attractive prey items [51,52]; whereas Antarctic soft corals and ascidians are instead highly energetic, attractive prey accounting for 16 KJ·g−1 and 15 KJ·g−1 DW (dry weight), respectively [53,54]. Assay diets in the current study were designed in order to obtain ranges of nutritional content (4.75–19 KJ·g−1 DW) ecologically reasonable and realistic according to the literature (to the mentioned analyses). The five types of isolated metabolites tested: wax esters (1–2), meridianins A–G (3–9), rossinone B (10), 5α(H)-cholestan-3-one (11) and glassponsine (12) were incorporated separately in the artificial foods via Phytoplan®, each at three different quantities: 2, 1, and 0.5 mg (from now on expressed in concentration: 10, 5, 2.5 mg·g−1TOTAL·DW). Compounds were dissolved in appropriate solvent carrier (either diethyl ether or methanol) to totally wet the dry components (food stimulant + sand), and the solvent was then evaporated, resulting in a uniform coating of the compound on the powdered food concentrate and sand prior to being added to the alginate aqueous mix. The new metabolite, glassponsine (12), was moreover assayed at its natural tissue concentration in preliminary tests, (2.34 mg·g−1 [29]; Table 1). The three assay compound concentrations: [Conc] = 10, 5, 2.5 mg·g−1TOTAL·DW, were chosen based on 5 mg·g−1 being the active concentration in preliminary trials and previous studies [21,25,26,27]. Control food pearls were prepared identically but without including testing compounds, adding an equal volume of solvent alone. The diverse mixtures obtained were introduced into a syringe and added drop-wise to a 0.09 M (1%) CaCl2 (Calcic® of Kit Sferificacion®) water solution, where they gelatinized (i.e., polymerized) forming spheroid pearls of approximately 2.5 mm in diameter. The relative quantity of the ingredients required to produce a whole set of untreated control pearls for a single experiment was: 3 mL alginate solution + 200/100/50 mg Phytoplan® + 0/100/150 mg sand respectively (200 mg dry components). This amount guarantees the formation of 150 pearls (15 replicates × 10 pearls per replicate) as well as extra pearls for pilot trials investigating feeding-unrelated changes to the pearls (see next subsection). For compound-treated pearls, the three diet types also included each of the compounds incorporated at the three different concentrations. Thus, each of the five compound types (1–2, 3–9, 10–12) at three different concentrations ([Conc]'s), was separately incorporated in the three diets of different nutritive content, resulting in nine assays per metabolite (3 [Conc]'s × 3 diets), and a total of 45 experiments for the whole study (9 assays × 5 compounds; see Table 4; Figure 1). Feeding experiments with small food pearls allows the measuring of "defense activity per pearl".
|Compounds Tested
|Composition of Artificially Prepared Diets (Feeding Pearls)
Compound (mg)/PhytoPlan (mg)/Sand (mg)/Alginate Solution (mL)
|[Conc] DWTOTAL (mg·g−1)
|[Conc] Volume (mg·mL−1)
|Wax esters mix (1–2)
Meridianins A–G mix (3–9)
Rossinone B (10)
5α(H)-cholestan-3-one (11)
Glassponsine (12)
Control and treatment pearls (containing isolated compounds) were visually distinguished in paired assays by adding different liquid tasteless food dyes (red and green) to the alginate mix before spherification in CaCl2 solution. Pilot trials confirmed that feeding preferences of Cheirimedon femoratus did not differ between colored and uncolored pearls (p = 0.47), or between red and green colored pearls (p = 0.47). Nevertheless, control and treatment food pearls were randomly swapped to green or red colorations throughout the experimentation period. Paired preference assays were also performed with control pearls of the three different diet types (200, 100 and 50 mg of Phytoplan®), in order to contrast the preferences and consumption rates of the amphipod towards more or less energy-rich diets.
3.5. Feeding-Preference Bioassays with Amphipods
Lyssianasid amphipods of the abundant, eurybathic Antarctic species Cheirimedon femoratus were chosen for feeding experiments for representing appropriate realistic generalist predators. The base protocol applied was described in [19], and it has been previously used in other studies testing isolated compounds [21,25,26,27]. These amphipods are voracious opportunistic feeders with a circumpolar distribution [55,56]. Thousands of individuals were captured by scuba diving in Port Foster Bay (Deception Island, South Shetland Archipelago: 62°59.369ʹ S, 60°33.424ʹ W) with fishing nets, between 2 and 7 m depth during the Antarctic cruise ACTIQUIM-3 (January 2011–March 2012). Once the experiments were completed, living specimens were returned to the sea. Amphipods were starved for 3 days while maintained in 8 L aquariums at the labs of the Spanish Base BAE "Gabriel de Castilla" (Deception Island) where the bioassays took place. Each assay consisted of 15 replicate containers filled with 500 mL of sea water and 15 amphipods, which were offered a simultaneous choice between 10 treatments (incorporating isolated compounds) and 10 control pearls (see Figure 1). When food items are small (<5 mm diameter) changes in mass due to consumption are negligible, and thus consumption or rejection is scored as the number of individual pellets [3]. The assays ended 5 h after food presentation, and the number of leftover pearls of each color (control or treatment) was then recorded. A food pearl was considered eaten when it was ingested up to remaining ~1/8 its original size (using a 1 mm2 mesh template). Since our feeding trials were short in time, mass autogenic alterations were avoided, and there was no need to run controls in the absence of amphipods for changes unrelated to consumption [57]. Uneaten or unused extra treatment pearls were assessed by TLC screenings to check for possible alterations related to chemical degradation or loss of the test compounds. TLC does not provide exact compound concentrations, but it may reveal a rough chemical profile of the metabolites present within a chemical extract/fraction, in situations when accessibility to more sophisticated equipment is limited (e.g., Antarctica). Since experimental timings were short (5 h), and Antarctic seawater temperature is very cold (≈1 °C), the modification/loss of organic products into the water column should be negligible.
3.6. Statistical Analysis
Differences in the quantity of ingested control and treated foods were calculated by counting the number of remaining uneaten pearls. Changes in the amount of the two foods in each container are not independent and have correlated errors. Each replicate is thus represented by a paired result yielding two sets of data (treatment and control). Since assumptions of normality and homogeneity of variances were not met, data were compared by non-parametric procedures applying Exact Wilcoxon Tests. The average ingestion rates for the three types of compound-free control diets holding different energetic content (made with 200, 100 and 50 mg of Phytoplan®) were calculated considering the data recorded for control feeding pearls (in 5 h exposure) in all the experiments performed of the study (45 assays in total). Data were evaluated for normality using Kolmogorov–Smirnov and Shapiro tests and for homogeneity of variance with Bartlett. Significant differences in the average ingestion rates in the three base control diets was contrasted by applying a one-way analysis of variance (ANOVA, α = 0.05), followed by a Tukey (HDS) post hoc test to determine two by two differences. The interaction between feeding deterrence and energetic content of assay foods was assessed statistically. Contingency tables 3 × 3 were constructed for each of the five compound types assessed (Wax esters 1–2, Meridianins 3–9, Rossinone 10, 5α(H)-cholestan-3-one 11, Glassponsine 12), with the two variables "Assay diet" and "Compound concentration". Assay diet had three categories, corresponding to the three base diets of different energetic content (pearls prepared with 200, 100 and 50 mg of Phytoplan®); while compound concentration included the three categories of product quantity (10, 5 and 2.5 mg·g−1 DWTOTAL) incorporated into the artificial foods. For each of the experiments, we calculated the average of the difference in consumption between paired control (compound-free) vs. treatment (with compound) feeding pearls within the 15 replicate tests, and further divided this value by the average ingestion rate of the 15 control pearl-based foods of that experiment. This value was then inserted in the corresponding contingency table as percentage: i.e., ∂PhytoPlan:[Conc] (see Supplementary Figure S1).
G-tests of independence with Williams's correction were then applied to determine whether the two categorical variables are associated with one another (Diet energetic content vs. Compound concentration), in relation with the bioactivity of each of the compound types assayed in the distinct conditions. The larger the G value (likelihood ratio) is, the greater is the probability (and level) of interaction/dependence between the two variables (and the smaller the p-value). The null hypothesis in the G-test is that the relative proportions of one variable are independent of the second variable, and significant tests (p < 0.05) reveal interference between variables.
All tests and most graphing material were performed on R-command software using R Studio, version v0.98.507, run with R statistics package, version 3.1.0 [58].
4. Conclusions
Antarctic invertebrates, and in general marine organisms, extensively use chemical defenses to control predation. Nevertheless, much needs to be learned about how antipredatory agents operate [2,5,17,18]. With this study, we showed that there is no relationship between the level of potency of a defensive chemical and the concentration in which it appears in the organisms' tissues. The most naturally abundant metabolites, meridianins and waxes, were respectively the most and the least active deterrents. Moreover, each compound may exhibit variable levels of repellency, and they may perform differently when mixed with distinct assay foods. The natural products that interacted the most with energetic content were those occurring in nature at higher concentrations, again meridianins and wax esters. The remaining metabolites (rossinone, 5α(H)-cholestan-3-one and glassponsine) require a threshold concentration to elicit feeding repellence, regardless of nutritional quality. The efficacy of a defensive product as repellent (in terms of minimal concentrations to afford activity), and the way it may interact with high energy foods is difficult to predict and seems to be characteristic of each compound type. Indeed, different types of compounds may interfere very uniquely with dietary components. Several authors have proposed that, even if it has not been sufficiently investigated, the way nutrients may mask the stimuli that elicit avoidance is by these components attaching to specific moieties of deterrent metabolites. Nutrient attractants might, in such a manner, compete for binding sites of taste receptors offsetting repellents' effect. Such receptors seem to be, indeed, highly conserved in different taxa, in terms of biochemical constituents, ([7] and references therein). An exciting issue for future research would be to elucidate the interactions between nutrients and defensive metabolites at the molecular level.
Supplementary Files
We thank M. Carbone and M. Gavagnin for corroborating the chemical structural data, and F. Castelluccio, M. Rodríguez-Arias, C. Angulo, J. Cristobo, B. Figuerola, J. Vázquez, J. Moles and S. Taboada for support and help in the lab. Thanks are due to "El Rey Moro" (P. Casanova) and L. Orlando for the artwork. We are also grateful to W. Arntz and the crew of R/V Polarstern, UTM (CSIC), "Las Palmas" and BAE "Gabriel de Castilla" crews for logistic support. First author is granted by the Postdoctoral program of Fundación Ramón Areces (Madrid, Spain). Funding was provided by the Spanish Government through three Antarctic projects (CGL/2004-03356/ANT, CGL2007-65453/ANT and CTM2010-17415/ANT).
Author Contributions
C. Avila and L. Núñez Pons collected the invertebrate samples. C. Avila leaded the project. The experimental design was elaborated by L. Núñez Pons. Amphipods for the tests were collected by L. Núñez Pons and C. Avila. Chemical extractions, metabolite purifications, experiments and results analysis were performed by L. Núñez Pons. The manuscript was written by L. Núñez Pons and C. Avila.
Conflicts of Interest
The authors declare no conflict of interest.
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Members of a local veterans group will place more than 200 cross-shaped markers on the grounds of Livonia's Nehasil Park Monday to mark the lives of Michigan soldiers who died in the Iraq and Afghanistan wars.
Two local chapters of Veterans For Peace, a national veterans organization with an anti-war message, are putting together the memorial, which they call "Arlington Michigan" -- a reference to Arlington National Cemetery.
The memorial has been held in various locations around the state for about a decade. This is the second year that Livonia's city council has allowed the memorial to set up in the park.
Steve Saelzler, 62, of Brownstown, is the coordinator for Chapter 74 of Veterans For Peace. He said the group doesn't bring anti-war signs to the memorial and avoids getting into ideology at the event, unless someone wants to have a discussion.
"We try to keep the politics out of it," Saelzler said. "It's just a reminder of the true costs of war and to commemorate or memorialize, in a way, those who have paid the ultimate price."
Besides organizing the annual memorial, members of the group also participate in parades and rallies throughout the year, like protesting against the military training at the School of the Americas.
Sometimes they hitch onto other organization's events, like so-called "healthcare not warfare" demonstrations or local anti-foreclosure actions. They also do political advocacy and outreach at local art fairs, which occasionally brings them in contact with both military recruiters and veterans' organizations that hold differing philosophies.
"We have a different mindset," Saelzler explained. "We're not against the military, [although] I personally do not see anything we should have been involved in since World War II. The price we keep paying in blood and treasure -- it's just crazy."
Veterans for Peace is a national organization that was founded in 1985 to oppose the Reagan administration's military intervention in Central America.
Chapter 74, which covers the southeast Michigan counties of Wayne, Monroe, Macomb and Oakland, formed in 1993 shortly after the first Iraq war. About 40 people are registered with the chapter and seven are active members. The membership at one time included a sizable number of World War II veterans, as well as individuals who fought in Korea and the Spanish Civil War, but now it's primarily composed of Vietnam Vets.
Saelzler spent a year in Vietnam's central highlands from '69 to '70 as a member of the Army's 4th Division. He saw combat, something he regrets.
"I was drafted ... and I didn't really have the courage to go to prison or stay away from my family in another country," he said. "So I did something I didn't really want to do."
Angry about the war, Saelzler shared his feelings with military brass by flouting rules and ended up spending the last month of his service in correctional custody back at Fort Knox. He managed to leave with an honorable discharge, but the Army made him sign a statement that he would never re-enlist.
He joined a group called Vietnam Veterans Against the War (VVAW) that engaged in a number of disruptive actions, including a brief occupation of a local office of the military contractor Honeywell in 1972.
Eventually -- caught up in the responsibilities of working a fulltime job at Chrysler and raising several adopted and foster children with his wife -- Saelzler drifted out of activism.
It wasn't until the United States got involved in the first Iraq War that he "woke up," he says, and started attending anti-war rallies again. At a 1992 rally against U.S. policy toward Iraq at Detroit's Central United Methodist Church, he ran into a man named Bruce Sanderson who was wearing a Veterans For Peace sweatshirt. Saelzler and a handful of other vets, some of whom had been active in VVAW, launched a Detroit chapter the following year.
"I was just hoping to feel better," Saelzler said of his anti-war activism. "It wasn't entirely a selfless act -- I wanted to help stop wars."
Saelzler said he still encounters a lot of disillusionment from the newer vets he meets who served in Iraq and Afghanistan, though it hasn't translated into very much anti-war activism in southeast Michigan.
But he says the wave of conflicts following Sept. 11 have only strengthened his own conviction of the influential role militarism plays in U.S. society.
"I believe there is an endless war philosophy going on -- a military-industrial complex powerhouse that's invested in keeping these conflicts going, he said, "and it's disproportionately the poor and disadvantaged they are calling to make these sacrifices."
Veterans For Peace will hold its memorial on Monday, May 28 from 9 a.m. to 6 p.m. at Livonia's Larry Nehasil Park. Chapter 74 meets once a month at the Swords Into Plowshares Gallery and Peace Center at 33 East Adams in Detroit. For more information visit the organization's website.


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Donald Trump evades taxes since 1995
The Republican candidate, Donald Trump declared 916 dollars loss in 1995 which allowed him to generate to avoid any type of federal tax revenues to 18 years.
As he unveiled the New York Times, said Trump in your 1995 tax return substantial tax deduction which allowed legally any kind of federal taxes.
The current candidate left in its path at the beginning of the 1990s during the mismanagement of three casinos in Atlantic City, the foray into the world of airlines and its ill-timed acquisition of the Plaza Hotel in Manhattan.
The tax laws are particularly advantageous for wealthy taxpayers, which allowed Trump to use that loss of 916 million dollars to cancel, according to tax specialists say.
The loss of $ 916 million dollars in 1995 which was the amount sufficient to eliminate more than $ 50 million dollars per year in revenue for 18 years.
Donald Trump evades taxes since 1995 Reviewed by Sonia Mar on 8:15 PM Rating:


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Microsoft Corporation on Monday announced the immediate availability of Revenge of Arcade, a new collection of classic arcade games for Windows 9x and NT 4.0. This latest collection--which includes Ms. Pac Man, Xevious, Rally-X, Mappy, and Motto, joins earlier classic releases such as Microsoft Arcade and Return of Microsoft Arcade. The games in this collection are authentic duplications of the early 1980's classics.
Hey, there hasn't been a decent game released since 1985 anyway. :) Enjoy
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Donald Trump is the only presidential candidate who seems to get that the vast majority of Americans want ILLEGAL ALIENS deported, none moreso than the Hispanic, Asian and other naturalized citizens who endured years of red tape to become Real Americans. He wants to use Ike Eisenhower's approach to deportation.
Liberals are revising this historic event. To inoculate readers against their attempts to fictionalize this history -- just as they have everything from the McCarthy hearings to climate change -- I offer some research on this. Already I have to correct Fox News Latino -- Rupert Murdoch's attempt to pander to Spanish-speaking people.
From Fox News:
Donald Trump said that if he were to become president of the United States, part of his immigration policy would be modeled directly after a 1950s policy called "Operation Wetback."
In an interview published online for 60 Minutes Overtime, Trump told CBS anchor and correspondent Scott Pelley that his immigration plan to deport all undocumented immigrants in the country and allow them to come in on a per-case basis can work because President Dwight D. Eisenhower did it more than half a century ago.
"We are rounding them up in a very humane way, in a very nice way, and they're going to be happy because they want to be legalized," Trump explained in the portion of his 60 Minutes interview that did not make air.
Trump went on to defend his policy by citing Eisenhower's 1954 immigration policy, Operation Wetback, which deported roughly 1 million undocumented immigrants to Mexico.
The policy, named after an offensive slur toward Hispanics, expelled Mexican nationals and U.S. citizens and transported them deep into Mexico so as to limit their chance of ever attempting to make it back into the United States, according to Huffington Post Latino Voices.
"It is practical, it's going to work," Trump told Pelley. "You look back in the 1950s, you look back at the Eisenhower administration; take a look at what they did and it worked."Now anytime you quote Huffington Post, you spread garbage.
Mexico and the United States reached an agreement in World War II to send Mexican workers north under the Bracero program. However, that program collapsed due to corruption. Any time you deal with the Mexican government, you have corruption -- not that ours is all that goody two-shoes either.
From the Texas State Historical Association:
Increasing grievances from various Mexican officials in the United States and Mexico prompted the Mexican government to rescind the bracero agreement and cease the export of Mexican workers. The United States Immigration Service, under pressure from various agricultural groups, retaliated against Mexico in 1951 by allowing thousands of illegals to cross the border, arresting them, and turning them over to the Texas Employment Commission, which delivered them to work for various grower groups in Texas and elsewhere. Over the long term, this action by the federal government, in violation of immigration laws and the agreement with Mexico, caused new problems for Texas. Between 1944 and 1954, "the decade of the wetback." the number of illegal aliens coming from Mexico increased by 6,000 percent. It is estimated that in 1954 before Operation Wetback got under way, more than a million workers had crossed the Rio Grande illegally. Cheap labor displaced native agricultural workers, and increased violation of labor laws and discrimination encouraged criminality, disease, and illiteracy. According to a study conducted in 1950 by the President's Commission on Migratory Labor in Texas, the Rio Grande valley cotton growers were paying approximately half of the wages paid elsewhere in Texas. In 1953 a McAllen newspaper clamored for justice in view of continuing criminal activities by "wetbacks."Sound familiar?
And it was Mexico -- not the United States -- that ended the deal arbitrarily. That led to the rounding up and deportation of illegal aliens. For every one deported, seven or more self-deported. The illegal alien population fell 95% in the 1950s.
Interestingly, worker wages and the standard of living for Americans rose dramatically in the 1960s as we suffered "affluenza" -- a liberal-minted malady for the middle class having "too much."
Mexico has draconian immigration laws. It jailed without bond or trial for six months a Marine who strayed across its borders.
Trump is the only one standing up to them. Either the Republicans stand up to them or we get a third party.


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Welcome to Andersonville Elementary
Our Mission
Guiding, Reaching, and Supporting all students to be their best!
Our Vision
Our motto: " We will do nothing less than our best"
Andersonville Elementary School's mission is to guide, reach and support all students to achieve their personal best. We consider these our three pillars of success. At AES, we have been astonished at how well these three pillars have fit into the programs we offer. For the pillar of guiding we have identified and implemented programs such as breakfast buddies, after school tutoring, school wide behavior incentive programs, and daily assemblies that remind each student to put forth nothing less than their best. AES sees the pillar of reaching as extending our vision to our community. Our school offers parent training, community events, our partnership with the local church, and a Tiger Talk newsletter to communicate the positive things going on in our school. The pillar of support was another area of strength for our school community. We offer after-school tutoring programs, a daily enrichment block, additional academic support, and various programs to help our families in need.
None of our success would be possible without the dedication of the entire school staff! The teachers at Andersonville are both professionally and personally committed to the success of each and every student.


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(1835–1925) soldier and chaplain with the Union army (1859–64), born in New York City. After the Civil War he played a pivotal role in library development, a career he undertook after more than twenty years as a clergyman.
Subjects: Warfare and Defence.
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- Description
Product Overview:
Availability: Ships in 2 - 3 weeks Dimensions: 4' long Weight: 35 lbs. Assembly: No Assembly Required Collection: Royal Patio Pine Furniture Warranty: 20 Year Residential and 5 Year Commercial Limited Warranty Handcrafted In: Pennsylvania, USA Reviews:
This 4-foot wooden patio bench features Amish craftsmanship and treated wood construction for durability. This 4-foot wooden bench with a backrest may be the perfect bench for your patio or outdoor table. Constructed of pressure-treated Southern yellow pine, this item is designed to last for years with minimal or no maintenance.
This wood shop uses Number 1 grade wood and stainless steel screws to craft this beautiful patio bench and finish it to the highest quality standards with attention to detail and style. Each outdoor bench is created to bring you style and durability and help you enjoy years full of sunny days and relaxed evenings in your favorite outdoor setting.
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Collection Royal Patio Pine Furniture Warranty 20 Year Residential and 5 Year Commercial Limited Warranty Assembly No Assembly Required Handcrafted In Pennsylvania, USA Material Wood Wood Pine Wood Product Dimensions 4' long Weight 35 Ships in 2 - 3 weeks
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Some of the banks in Greenville, South Carolina include Bank of America, Wells Fargo and PNC Bank. There are also smaller banking and credit union branches, such as Regions Bank, Woodforest National Bank, SunTrust and TD Bank.Continue Reading
Bank of America is one of the top banks located in Greenville, South Carolina. It offers the same types of accounts and banking services as other Bank of America branches, including checking and savings accounts, IRAs, bonds and loans. Some of its checking accounts include Core Checking, Core Checking for Students and Interest Checking. The bank also has the Regular Savings account and Rewards Money Market Savings account.
Another bank is Wells Fargo, offering multiple checking and savings accounts. Its Everyday Checking accounts include overdraft protection, depositing checks from a mobile phone and unlimited check writing. There is also a PMA Package account, Wells Fargo at Work Checking account and Preferred Package account. All bank accounts come with online bill pay and mobile banking.
Woodforest National Bank is one of the smaller banks serving Greenville. It has a standard Woodforest Checking account, Second Chance Checking and Choice Checking account. All checking accounts at this bank include overdraft protection, online banking and bill pay, mobile banking and the option to have a savings account.Learn more about Banks


text: This simple, crushed berry limeade is made in a few minutes, and is the perfect way to cool down on a hot summers day. Use frozen berries, defrosted in the fridge, in place of fresh if you wish. If using agave nectar, this recipe is also vegan-friendly. Put the agave nectar (or honey), lime juice and berries together in a serving jug. Muddle (mash) with a wooden spoon handle to crush the berries. When well mixed, top up the jug with the chilled water. Pour into ice-filled glasses to serve. See more Drink recipes


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The Rev. Martin Luther King announced in a 1963 speech he was "…Proud to be maladjusted." There are certain realities, he argued, we should not entertain and need not adjust to as people and leaders. I have reflected on my own call to action this year and find, in Dr. King's slight twist on the word, a surprisingly liberating doorway for the New Year. Our current business world seems to require high levels of adjustment—research indicates upwards of 65% of leaders fall somewhere in the Reactive/Conventional or Socialized stage of development. It seems obvious we need more creative maladjustment! In this article I will discuss illusion, passion, sensuality, lovemaking, and finding the Essential in our work. I will suggest all play a role in performing our work at the highest level and can be exercised in seven practical actions. A good round number. I hope I now have your attention.
Eight hundred years ago Hafiz compared our journey to a desert caravan where the challenge was to discern what was real from what was mirage. Sitting here in my office, this seems straightforward. Apparently, trying to stay alive in a barren desert is an altogether different story. Trying to find what is true and essential in organizational life is daunting as well. Hafiz' perspective:
It has not rained light for many days,
The wells in most eyes look
Thus friends are not easy to find
In this barren
Where most everyone has become ill
From guarding
On this primal caravan
Careers and cities can appear real in this
Desert heat,
But I say to my close ones,
Don't get lost in them,
It has not rained light there for days.
Look, most everyone is diseased
From 'making love' to
(The Gift: Poems by Hafiz, the Great Sufi Master. Daniel Ladinski, editor.)
Let's begin by coming to terms with how marvelously easy it is to lose our way. We begin with humility. "…It has not rained light for many days…" I re-learned something the hard way last year: when I am weary and exhausted, stressed from an overwhelming set of priorities or secretly concerned that I may not measure up to the demands placed on me, my brain doesn't work well. I see things that are not there; I make up stories and treat as real something that is completely imaginary. I worry about things I cannot control and battle enemies that appear to be "out there" but are really "in here." I lose the ability to clearly tell the difference. The heat of my energy-demanding life, the torrid landscape of uncertainty, complexity, and endless demands on my time and calendar all take an invisible and cumulative toll. I feel thirsty for light.
• Action: Become strategically unavailable. I have purposed to build in time to breathe deeply and cool down. I suggest you find room to breathe, and travel more lightly, as well. Drought-stricken eyes are caused by an endless diet of problems and things gone wrong. Problems we take personally. I will find and pursue possibilities that awaken others and me so that we each have a more enjoyable and meaningful trip. I will learn how to rain light, a vital leadership competency not often listed in leadership journals.
In an illusionary world, where a mirage can make real what is not, we may need resiliency and resolve to reach our destination. In the interim, we live primarily with uncertainty and with what David Whyte calls "…The bitter unwanted passion of our sure defeat." Under this kind of pressure we can lapse into categorizing others around us friend or foe, ally, or enemy. These are not useful categories except on the wartime field of battle. Yet I have seen several instances where colleagues and I fell into this mindset: If you see the world differently than we do you are not one of us. You are with "them." Friends are hard to find in this barren place.
When complexity, pace of change, and scarcity of resources simultaneously escalate, the organizational world can become spiritually and emotionally barren. Sound bites pass as dialogue and heaving opinions back and forth passes for learning from one another. We want to be right more than effective. It is easy to see where we could feel justified in excluding those who seem different. We have simply adjusted to the norm of separation. We have adjusted to the mirage of separate and competing interests. We have fallen into the bleak, un-awakened landscape of believing that in order to get what I want you must lose something. Each time I am successful taking from you I become more vulnerable myself. I know you could do the same to me. My defenses must therefore be made stronger. On it goes. I personally have done these things even while righteously proclaiming I want to be creatively maladjusted. Welcome to the citadel of my life!
• Action: Cultivate friendships and new partnerships with others who may appear to see their world differently than you do but who are undoubtedly cut from the same cloth as you and me. "…Thus friends are hard to find in this barren place…" I have purposed to find ways to connect with others I see as different. I have spent too many days and years as a lone sentinel standing over my organization, my field of study, my professional discipline, and other things I mistook for me. I have begun to see that when I mistake my business strategies, my politics, my ideology, my religion, my nation, my organization for myself, I then lose the ability to live with compassion in the world. I then become just one more man, defending one more mirage and fighting one more senseless battle. It is enough.
When I spend time and energy defending my position, my opinion, my career, my technical expertise, my functional area—virtually anything that has the word "my" in front of it—I am very likely guarding nothing. "…Most everyone has become ill from guarding nothing…"
Guarding is essentially a reactive action, i.e. I must be vigilant from a threat that always emerges "out there" and be ready to defend. Even when my action appears strong and tough, it is motivated by a desire to guard or defend (myself and what I have mistaken to be real). Then my action is compensatory at best. I am playing to not lose. It is impossible to move into elite level work from such a defensive stance.
• Action: Watch closely what you defend and guard and get curious when and why you defend it. Curiosity is often much better than judgment. I have purposed to defend much less this year than in any other year. I desire that my action be purpose-driven—moving others and me towards outcomes that matter to us all—and no longer in protecting and guarding my illusory and false sense of self. I will watch closely how often I reference "me" and "my," seeing these indicators as a relevant set of (creatively maladjusted) metrics in a world that claims to like metrics.
Consider the real possibility that core systems and structures in our lives may be mirages. "…Careers and cities can appear real in this intense desert heat…" When you are under considerable heat pay careful attention to what you think you see. Mirages abound! The small footnote in the torn page of my life is worth remembering here. Careers, cities, businesses appear and appear real when you're in them! There are, after all, traffic lights, buildings, and noise. There are customers, incentive plans, and performance evaluations, pay grades, promotions, and turnover. I hope the last item does not include me! It is only in perspective, gained by stepping back and breathing, that I can begin to entertain the possibility that much of what I have invested my life energy in is temporary, impermanent, and likely to be gone soon. If it was ever there at all! But this is a perspective gained from being outside the mirage looking at it, not from within it.
• Action: Remember: things are not as they appear. I purpose to act as if all things are malleable. I see what I am prepared to see. Problems or opportunities? My call and yours. Nothing is as fixed, dense and "given" as I think it is. Neither my customers' behavior and circumstances nor my own behavior and circumstances are as dense as I once thought. There is hope. Then again, I might be a mirage myself, but that's another article.
From his desert experiences Hafiz implies there is an abiding spiritual reality behind things, an un-changing truth behind the shimmering mirage. "…I say to my close ones, 'Don't get lost in them'…" No prayer beads, wailing, or sacrifice are required, just curiosity. The underlying Essence is not always easy to find and doesn't announce itself with lights and flags waving. Yet, asking, "What's really going on here" seems to invite its response. Learn to bring this "meaning making" power we have into your direct conversation with others. When done right, this will at times feel uncomfortable and awkward, and require vulnerability and courage on your part. Particularly in misunderstandings where tensions get triggered, don't get lost in meaningless positions and exaggerated arguments. Why defend gravity?
• Action: Find your own way of pursuing and describing the essence of the issues with which you are faced. Use your intuition or your gut to discern the small voice or gentle hand on the small of your back saying, "Don't get lost in them." I have purposed to listen for the open secret hidden in plain sight, there in the midst of conversations with those I live and work with. I have tasted a different kind of connection with others and have grown weary of settling for something less. Plus, drought stricken eyes don't look cool.
The complexity, pace, and pressure of the workplace today require an unprecedented level of emotional and behavioral resiliency. We have to hang in with each other for the long haul caravan. For leaders, this means we have to speak into the heart of others in ways we may never have before. We appeal to drought-stricken eyes with our own vulnerability and openness to new learning, our courage to share our struggles, uncertainties, and concerns even as we pursue outcomes that are important but seem impossible. As we take others aside and share what we know with them (instead of using them as order takers) we are implicitly calling out and speaking to the inherent brilliance of others as we see it. What could be more important?
• Action: Expand your skill set by learning how to appeal to the mind, hands, and heart of those you work alongside. Most of us appeal only to the mind: we want to be seen as relevant, rational, and right. A new slant on the Three R's. We speak in declarative statements that reveal our thinking and position on things. If we are with each other long enough, we can easily predict one another's viewpoints before we open our mouths. Put down the spreadsheet or your cash flow analysis and look out. I have laid mine aside too. I'll meet you in a different conversation.
The short story here is this: leadership is not only or even primarily a rational discipline. When done well, it awakens passions and emotions. When done at a high level, leadership combines elements of inward and outward strength, power, authority, touch, vulnerability, sensitivity, imagination, connection, intuitive knowing, and risky conversation. Leadership is, at times, very sensual. It can involve lovemaking of a highest order—devotion not just to good work and purposeful achievement but also to inevitable work, that which we came into life to do and to that Essential part of us that calls us to it. It is only in this larger context that we can see more clearly how easy it is to "…Become diseased from making love with nothing." A look at the headlines in any newspaper confirms how easily we can be duped into settling for something, or someone, less.
• Final Action: Love what is real. "Look, almost everyone is diseased from making love with nothing." Imagine the CNN headlines: American health care costs skyrocket—Defending and making love with nothing reach epidemic levels in the workplace! If you're considering a creatively maladjusted life you've got to appreciate the desert mystics of another time and era. Their love of the Divine was sensual; their ecstasy in union whole-body! Their connection with The Essential, Body-Consuming.
Mine is something less than this.
I have purposed to follow the thread of the Essential as it seeks expression in my life and work this year. I do not want to fall prey to fighting small battles over nothing and finding when I win I actually become smaller. I want to be decisively defeated by what is Essential. I will do my best to ask: What matters most here? What seems to want to happen here? What truth lies behind and fuels the frustration I see in others and myself?
May you see the inherent beauty in and come to trust your own maladjustment. Remember, we are not supposed to adjust to inequity, injustice, mediocrity, and deception. What are the additional things you know you are not supposed to adjust to this year? Join the creatively maladjusted community of leaders and those who work with them. We are a small but deceptively powerful group caravanning through a world in dire need of magic!


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Regarding Ethan Couch's sentence, does the judge not realize she is doing what the parents did for so many years? She is not holding him accountable for his actions. What is probation teaching him? Kids that age need structure and guidelines.
I cringe to think what he will be like after his probation. The judicial system has taught him that with wealth he can get away with killing people. What a travesty. Everyone has failed this boy.
— Terri Horn, Crowley


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Instantly hide an unattractive central heating essential with a radiator cover, because let's be honest electric radiators are rarely beautiful pieces of contemporary design. Nor do they have the charm of traditional cast-iron radiators. However, when disguised with clever modern radiator covers and smart cabinets, this essential feature of the modern home (no-one wants to lose their central heating!) can blend in with the rest of your decorating scheme.
Radiator covers can, in fact, be extremely practical: they hide intrusive, unsightly radiators, protect children from burning themselves, and some even provide extra storage in the form of an above-radiator shelf. No longer associated solely with traditional decor, these days there are a number of contemporary designer radiator covers, contemporary cabinets and stylish radiator grills that suit many up-to-date schemes from minimalist to retro modern. Not convinced? Just take a look at our selection of the best radiator covers you can buy now.
New Suffolk Radiator Cover
This petite radiator cover is the perfect fit for smaller rooms and radiators. The fresh painted white wood and clean lined design make an amicable addititon to modern coastal schemes. Painted MDF.
Dimensions: H80 W102 D18cm.
Glass Radiator Cover
Turn drab radiators into a modern feature with this stylish Adam Fire Surrounds glass radiator cover in black. Made of sleek black tempered glass, this cover slots effortlessly over a radiator to turn a functional feature into something truly eye-catching. It is perfect for revitalising any room without having to splash out on decorating. H64 W120 D3.5cm.
Forest Radiator Cover Cabinet
Cover an unattractive radiator with this fresh white painted cover with decorative diamond grill pattern. The clever design is adjustable, making it perfect for accommodating most radiator sizes and also making more of a statement; the generous size means it could pass as merely a console unit in a hallway – the perfect radiator cover up. Comes pre-cut to accommodate a skirting board, giving a flush to the wall finish. H81.7 W140-200 D19cm.
Unfinished MDF Radiator Cover
Plain MDF radiator covers such as this design can be great budget options. You will need to prime and paint this radiator cabinet, but this is still more likely to work out cheaper than buying an equivalent ready-finished version. This one has a smart double-panelled grille. Unfinished MDF. H90 W120 D20cm.
Medium Unfinished Cambridge Radiator Cover
A great budget radiator cover with a double arched grille design that will hide unsightly radiators. Unfinished MDF, allowing the option to paint to match a decorating scheme. H90 W119.8 D20cm.
Cambridge Large White Painted Radiator Cover
This radiator cover from the Cambridge range provides a practical solution for concealing old or unfashionable radiators around the home. The MDF design also offers extra surface space to display belongings and protection against hot surfaces. H90 W171 D20cm.
Victorian-style Adjustable and Unfinished Budget Radiator Cover
This three-panel adjustable radiator cover is suitable for wall-mounted, flat-panel radiators. Attractive Victorian styling with three-panel grille – simply prime and paint to match your decor. With traditional good looks and a large ledge, this classic radiator cover tops our wish list. Take advantage of it's size by using it in the dining room instead of a sideboard.Quick and easy to fit. Unfinished MDF. H93.6 W97-142 D23.5cm.
Mayfair White Painted Radiator Cover
This white painted radiator cover is simply designed to suit any decorating scheme. The central grille is complemented by a curved skirting. This radiator cover is also available in two other widths. H81.5 W111.5 D19cm.
Adam Unfinished MDF Radiator Cover
Plain MDF radiator covers such as this design can be great budget options. You will need to prime and paint this radiator cabinet, but this is still more likely to work out cheaper than buying an equivalent ready-finished version. This one has a smart double-panelled grille. Unfinished MDF. H90 W120 D20cm.
CASA Moroccan Radiator Covers and Console Tables
This modern Moroccan-style radiator manages to be both contemporary and classic and will fill any living space with eastern promise. The intricate pattern is laser cut out of premium zinc metal and finished with white satin specialised paint. H65 W70cm.
Modern Radiator Covers